Jak kinase inhibitor compounds for treatment of respiratory disease

ABSTRACT

The invention provides compounds of formula (I):wherein X isand the variables are defined in the specification, or a pharmaceutically-acceptable salt thereof, that are useful as JAK kinase inhibitors. The invention also provides pharmaceutical compositions comprising such compounds, methods of using such compounds to treat respiratory diseases, and processes and intermediates useful for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/250,113, filed on Nov. 3, 2015; the entire disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The invention is directed to diamino compounds useful as JAK kinaseinhibitors. The invention is also directed to pharmaceuticalcompositions comprising such compounds, methods of using such compoundsto treat respiratory diseases, and processes and intermediates usefulfor preparing such compounds.

State of the Art

Asthma is a chronic disease of the airways for which there are nopreventions or cures. The disease is characterized by inflammation,fibrosis, hyperresponsiveness, and remodeling of the airways, all ofwhich contribute to airflow limitation. An estimated 300 million peopleworldwide suffer from asthma and it is estimated that the number ofpeople with asthma will grow by more than 100 million by 2025. In theUnited States, asthma afflicts about 6% to 8% of the population, makingit one of the most common chronic diseases in the country. Although mostpatients can achieve control of asthma symptoms with the use of inhaledcorticosteroids that may be combined with a leukotriene modifier and/ora long acting beta agonist, there remains a subset of patients withsevere asthma whose disease is not controlled by conventional therapies.Severe persistent asthma is defined as disease that remains uncontrolledon high doses of inhaled corticosteroids. While severe asthmatics areestimated to account for approximately 5% of all asthma sufferers, theyhave a high risk of morbidity and mortality and are responsible for adisproportionate share of health care resource utilization amongasthmatics. There remains a need for novel therapies to treat thesepatients.

Cytokines are intercellular signaling molecules which includechemokines, interferons, interleukins, lymphokines, and tumour necrosisfactor. Cytokines are critical for normal cell growth andimmunoregulation but also drive immune-mediated diseases and contributeto the growth of malignant cells. Elevated levels of many cytokines havebeen implicated in the pathology of asthma inflammation. For example,antibody-based therapies targeted at interleukins (IL)-5, and 13 havebeen shown to provide clinical benefit in subsets of severe asthmapatients. Among the cytokines implicated in asthma inflammation, manyact through signaling pathways dependent upon the Janus family oftyrosine kinases (JAKs), which signal through the Signal Transducer andActivator of Transcription (STAT) family of transcription factors.Cytokines implicated in asthma inflammation which signal through theJAK-STAT pathway include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-11,IL-13, IL-23, IL-31, IL-27, thymic stromal lymphopoietin (TSLP),interferon-γ (IFNγ) and granulocyte-macrophage colony-stimulating factor(GM-CSF).

The JAK family comprises four members, JAK1, JAK2, JAK3, and tyrosinekinase 2 (TYK2). Binding of cytokine to a JAK-dependent cytokinereceptor induces receptor dimerization which results in phosphorylationof tyrosine residues on the JAK kinase, effecting JAK activation.Phosphorylated JAKs, in turn, bind and phosphorylate various STATproteins which dimerize, internalize in the cell nucleus and directlymodulate gene transcription, leading, among other effects, to thedownstream effects associated with inflammatory disease. The JAKsusually associate with cytokine receptors in pairs as homodimers orheterodimers. Specific cytokines are associated with specific JAKpairings. Each of the four members of the JAK family is implicated inthe signaling of at least one of the cytokines associated with asthmainflammation. Consequently, a chemical inhibitor with pan-activityagainst all members of the JAK family could modulate a broad range ofpro-inflammatory pathways that contribute to severe asthma.

However, the broad anti-inflammatory effect of such inhibitors couldsuppress normal immune cell function, potentially leading to increasedrisk of infection. Evidence of increased infection risk has beenobserved with the JAK inhibitor tofacitinib, which is dosed orally forthe treatment of rheumatoid arthritis. In asthma, inflammation islocalized to the respiratory tract. Inflammation of the airways ischaracteristic of other respiratory diseases in addition to asthma.Chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF),pneumonitis, interstitial lung diseases (including idiopathic pulmonaryfibrosis), acute lung injury, acute respiratory distress syndrome,bronchitis, emphysema, and bronchiolitis obliterans are also respiratorytract diseases in which the pathophysiology is believed to be related toJAK-signaling cytokines. Local administration of a JAK inhibitor to thelungs by inhalation offers the potential to be therapeuticallyefficacious by delivering a potent anti-cytokine agent directly to thesite of action, limiting systemic exposure and therefore limiting thepotential for adverse systemic immunosuppression. The need remains for apotent JAK inhibitor suitable for local administration to the lungs fortreatment of respiratory disease.

SUMMARY OF THE INVENTION

In one aspect, the invention provides novel compounds having activity asJAK kinase inhibitors.

Accordingly, the invention provides a compound of formula (I):

wherein:

X is a group of formula (II):

n is 0 or 1;

R¹ is hydrogen or C₁₋₃alkyl;

R² is hydrogen or C₁₋₃alkyl;

R³ is hydrogen or C₁₋₃alkyl;

-   -   or R² and R³ taken together form C₂₋₄alkylene;    -   or, when n is 1, R³ is selected from hydrogen, —OH, —OC₁₋₃alkyl,        halo, —C(O)OC₁₋₃alkyl, and C₁₋₃alkyl, wherein C₁₋₃alkyl is        optionally substituted with —OH;

R⁴ is hydrogen or C₁₋₃alkyl;

R⁵ is selected from hydrogen, C₁₋₃alkyl, —C(O)OC₁₋₃alkyl, and phenyl;

-   -   or when n is 1, R² and R⁵ taken together form C₁₋₃alkylene;

R⁶ is hydrogen or C₁₋₃alkyl;

R⁷ is hydrogen or C₁₋₃alkyl,

-   -   or when n is 0, R² and R⁷ taken together form C₁₋₃alkylene, or        R⁴ and R⁷ taken together form C₂₋₄alkylene or        C₁alkylene-O—C₂alkylene;    -   or when n is 1, R² and R⁷ taken together form C₂alkylene,        optionally substituted with C₁₋₃alkyl or R^(x),        -   or R⁴ and R⁷ taken together form C₁₋₃alkylene or            —O—C₂alkylene;

R⁸ is selected from

-   -   (a) hydrogen,    -   (b) methyl, optionally substituted with —CN, phenyl or        C₃₋₆cycloalkyl; (c) C₂₋₆alkyl, wherein C₂₋₆alkyl is optionally        substituted with one or two substituents selected from —OH,        —OC₁₋₃alkyl, —CN, —SC₁₋₃alkyl, phenyl, C₃₋₆cycloalkyl, halo, and        optionally, in addition with two substituents on a single carbon        atom taken together to form C₂₋₃alkylene;    -   (d) C₃₋₆cycloalkyl, wherein C₃₋₆cycloalkyl is optionally        substituted with —OH, —CN, —OC₁₋₃alkyl, or C₁₋₃alkyl, wherein        C₁₋₃alkyl is optionally substituted with —OC₁₋₃alkyl or with one        or two halo,    -   (e) oxetanyl,    -   (f) tetrahydropyranyl,    -   (g) tetrahydrothiophenyl 1,1-dioxide, and    -   (h) phenyl,

or R⁷ and R⁸ taken together form C₃₋₅alkylene orC₂alkylene-O—C₂alkylene; wherein C₃₋₅alkylene is optionally substitutedwith one or two R^(x);

R^(x) is selected from —OH, —CN, —OC₁₋₃alkyl, halo, phenyl, andC₁₋₃alkyl wherein C₁₋₃alkyl is optionally substituted with —OC₁₋₃alkylor —OH, or

two substituents R^(x) taken together form C₁₋₅alkylene or —CH₂OCH₂—,

or when n is 1 and R² and R⁷ taken together form C₂alkylene, R⁴ and asubstituent R^(x) on C₂alkylene taken together form C₂alkylene;

provided that two substituents R^(x) on the same carbon atom are notboth fluoro, and

provided that when R^(x) is attached to a carbon atom adjacent to anitrogen atom, R^(x) is not —OH, —OC₁₋₃alkyl, or halo;

or a pharmaceutically-acceptable salt thereof.

As used hereinafter, the phrase “compound of formula (I)” means acompound of formula (I) or a pharmaceutically acceptable salt thereof;i.e., this phrase means a compound of formula (I) in free base form orin a pharmaceutically acceptable salt form unless otherwise indicated.

The invention also provides a pharmaceutical composition comprising acompound of the invention and a pharmaceutically-acceptable carrier.

In another aspect, the invention provides a particular compound offormula (I) as a crystalline free base hydrate. The crystalline hydrateof5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolhas been found to have a melting temperature in the range of about 206°C. to about 216° C., typically between about 209° C. and about 214° C.,a decomposition onset at about 245° C., and to exhibit weight changes ofless than about 0.12% when exposed to a range of relative humiditybetween about 5% and about 90% at room temperature.

The invention also provides a method of treating respiratory disease, inparticular, asthma, in a mammal, the method comprising administering tothe mammal a therapeutically effective amount of a compound or of apharmaceutical composition of the invention. In separate and distinctaspects, the invention also provides synthetic processes andintermediates described herein, which are useful for preparing compoundsof the invention.

The invention also provides a compound of the invention as describedherein for use in medical therapy, as well as the use of a compound ofthe invention in the manufacture of a formulation or medicament fortreating respiratory disease in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the present invention are illustrated by reference tothe accompanying drawings.

FIG. 1 shows a powder x-ray diffraction (PXRD) pattern of thecrystalline hydrate of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol.

FIG. 2 shows a differential scanning calorimetry (DSC) thermogram of thecrystalline hydrate of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol.

FIG. 3 shows a thermal gravimetric analysis (TGA) plot of thecrystalline hydrate of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol.

FIG. 4 shows a dynamic moisture sorption (DMS) isotherm of thecrystalline hydrate of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolobserved at a temperature of about 25° C.

DETAILED DESCRIPTION OF THE INVENTION

Among other aspects, the invention provides JAK kinase inhibitors offormula (I), pharmaceutically-acceptable salts thereof, andintermediates for the preparation thereof. The following substituentsand values are intended to provide representative examples of variousaspects of this invention. These representative values are intended tofurther define such aspects and are not intended to exclude other valuesor limit the scope of the invention.

In a specific aspect X is a group of formula (II):

In another specific aspect, X is selected from

wherein the variables R⁴, R⁵, R⁷, R⁸, and R^(x) are defined as informula (I) or are defined as hereinbelow, R³ is hydrogen or C₁₋₃alkyl,and R^(3a) is selected from hydrogen, —OH, —OC₁₋₃alkyl, halo,—C(O)OC₁₋₃alkyl, and C₁₋₃alkyl, wherein C₁₋₃alkyl is optionallysubstituted with —OH. In another aspect, R^(3a) is selected fromhydrogen, —OH, —OC₁₋₃alkyl, halo, —C(O)OC₁₋₃alkyl, and C₁₋₃alkyl,

In yet another aspect, X is selected from:

where the variables R^(3a), R⁷, and R⁸ are defined as describedimmediately above or where, in particular, R^(3a) is halo and thepyrrolidine ring of

is optionally substituted with C₁₋₃alkyl.

In yet another aspect, X is selected from:

wherein:

R⁷ is C₁₋₃alkyl; and

R⁸ is selected from hydrogen, methyl, C₂₋₄alkyl, C₃₋₄cycloalkyl, and

In a specific aspect n is 0 or 1. In another specific aspect, n is 0. Inyet another specific aspect, n is 1.

In a specific aspect, R¹ is hydrogen or C₁₋₃alkyl. In another specificaspect, R¹ is hydrogen.

In a specific aspect, R² is hydrogen or C₁₋₃alkyl. In another specificaspect, R² is hydrogen.

In a specific aspect, R³ is hydrogen or C₁₋₃alkyl; or R² and R³ takentogether form C₂₋₄alkylene; or, when n is 1, R³ is selected fromhydrogen, —OH, —OC₁₋₃alkyl, halo, —C(O)OC₁₋₃alkyl, and C₁₋₃alkyl,wherein C₁₋₃alkyl is optionally substituted with —OH;

In another specific aspect, R³ is hydrogen or C₁₋₃alkyl; or, when n is1, R³ is selected from hydrogen, —OH, —OC₁₋₃alkyl, halo,—C(O)OC₁₋₃alkyl, and C₁₋₃alkyl;

In a specific aspect, R² and R³ taken together form C₃alkylene;

Specific values of R³ include hydrogen, —CH₃, —OH, —CH₂OH, and fluoro.

In a specific aspect, R⁴ is hydrogen or C₁₋₃alkyl. In another specificaspect, R⁴ is hydrogen.

In a specific aspect, R⁵ is selected from hydrogen, C₁₋₃alkyl,—C(O)OC₁₋₃alkyl, and phenyl, or when n is 1, R² and R⁵ taken togetherform C₁₋₃alkylene.

In another specific aspect, R² and R⁵ taken together form C₁alkylene

In another specific aspect, R⁵ is hydrogen or C₁₋₃alkyl. In yet anotherspecific aspect, R⁵ is hydrogen

In a specific aspect, R⁶ is hydrogen or C₁₋₃alkyl. In another specificaspect, R⁶ is hydrogen.

In a specific aspect, R⁷ is hydrogen or C₁₋₃alkyl or when n is 0, R² andR⁷ taken together form C₁₋₃alkylene, or R⁴ and R⁷ taken together formC₂₋₄alkylene or C₁alkylene-O—C₂alkylene; or when n is 1, R² and R⁷ takentogether form C₂alkylene, optionally substituted with C₁₋₃alkyl, or R⁴and R⁷ taken together form C₁₋₃alkylene or —O—C₂alkylene. In anotherspecific aspect, when n is 1, R⁴ and R⁷ taken together form—O—C₂alkylene.

In another specific aspect, R⁷ is hydrogen or C₁₋₃alkyl.

In a specific aspect, R⁸ is selected from (a) hydrogen, (b) methyl,optionally substituted with —CN, phenyl or C₃₋₆cycloalkyl; (c)C₂₋₆alkyl, wherein C₂₋₆alkyl is optionally substituted with one or twosubstituents selected from —OH, —OC₁₋₃alkyl, —CN, —SC₁₋₃alkyl, phenyl,C₃₋₆cycloalkyl, halo, and optionally, in addition with two substituentson a single carbon atom taken together to form C₂₋₃alkylene; (d)C₃₋₆cycloalkyl, wherein C₃₋₆cycloalkyl is optionally substituted with—OH, —CN, —OC₁₋₃alkyl, or C₁₋₃alkyl, wherein C₁₋₃alkyl is optionallysubstituted with —OC₁₋₃alkyl or with one or two halo, (e) oxetanyl, (f)tetrahydropyranyl, (g) tetrahydrothiophenyl 1,1-dioxide, and (h) phenyl;or R⁷ and R⁸ taken together form C₃₋₅alkylene orC₂alkylene-O—C₂alkylene.

In another specific aspect, R⁸ is selected from (a) hydrogen, (b)methyl, optionally substituted with C₃₋₆cycloalkyl; (c) C₂₋₄alkyl,wherein C₂₋₄alkyl is optionally substituted with one substituentselected from —OH, —OC₁₋₃alkyl, —CN, —SC₁₋₃alkyl, C₃₋₄cycloalkyl, andhalo and optionally, in addition, with two substituents on a singlecarbon atom taken together to form C₂alkylene; (d) C₃₋₄cycloalkyl,wherein C₃₋₄cycloalkyl is optionally substituted with —OH, —CN,—OC₁₋₃alkyl, or C₁₋₃alkyl, wherein C₁₋₃alkyl is optionally substitutedwith —OC₁₋₃alkyl or with one or two halo; (e) oxetanyl; (f)tetrahydropyranyl; and (g) tetrahydrothiophenyl 1,1-dioxide.

Specific values of R⁸ include hydrogen, —CH₃, —C₂H₅, isopropyl,cyclopropyl, cyclobutyl, —CH(CH₃)C₂H₅, —(CH₂)₂CN, —CH₂CH₂F,—CH₂isopropyl —CH₂cyclopropyl, —(CH₂)₂OH, (CH₂)₂₋₃OCH₃, —(CH₂)₂₋₃SCH₃,—(CH₂)₂CH(CH₃)SCH₃, tetrahydropyran-4-yl, pyridin-4-yl,

In other specific aspects, R⁸ is selected from hydrogen, methyl,C₂₋₄alkyl, C₃₋₄cycloalkyl, and

and hydrogen, methyl, C₂₋₄alkyl and

In yet another specific aspect, R⁸ is selected from hydrogen, methyl,C₂₋₄alkyl, and C₃₋₄cycloalkyl, or hydrogen, methyl, C₂₋₄alkyl andC₃cycloalkyl.

In a certain aspect, the invention provides compounds of formula (III):

wherein the variable R⁸ is as defined herein.

In another aspect, the invention provides compounds of formula (IV):

wherein the variable R⁸ is as defined herein.

In yet another aspect, the invention provides compounds of formula (V):

wherein the variables R⁷ and R⁸ are as defined herein.

In another aspect, the invention provides a compound selected from thefollowing compounds

5-ethyl-2-fluoro-4-(3-(5-(1-methylazetidin-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,

4-(3-(5-(azetidin-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol,

5-ethyl-2-fluoro-4-(3-(5-(1-isopropylazetidin-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,

4-(3-(5-(1-(sec-butyl)azetidin-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol,

4-(3-(5-(1-cyclopropylazetidin-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol,

5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,

4-(3-(5-(2-(dimethylamino)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol,

5-ethyl-2-fluoro-4-(3-(5-(2-((3-methoxycyclobutyl)amino)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,

5-ethyl-4-(3-(5-(2-(ethyl(methyl)amino)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-2-fluorophenol,

4-(3-(5-(2-(sec-butyl(methyl)amino)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol,

(S)-5-ethyl-2-fluoro-4-(3-(5-((1-methylpyrrolidin-2-yl)methyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,

4-(3-(5-(3-(dimethylamino)-2-fluoropropyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol,

(S)-5-ethyl-2-fluoro-4-(3-(5-(morpholin-3-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,

(R)-5-ethyl-2-fluoro-4-(3-(5-(morpholin-3-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,

(S)-5-ethyl-2-fluoro-4-(3-(5-(2-(2-methylpyrrolidin-1-yl)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,

and pharmaceutically-acceptable salts thereof.

In one aspect, the invention provides the compounds of Examples 1-18 andTables 1-19 below.

In a specific aspect, the invention provides the compounds 3-19, 3-28,and 3-29, disclosed in Table 3 that are not included in formula (I).

Chemical structures are named herein according to IUPAC conventions asimplemented in ChemDraw software (PerkinElmer, Inc., Cambridge, Mass.).For example, the compound of Example 1:

is designated as5-ethyl-2-fluoro-4-(3-(5-(1-methylazetidin-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol.

Furthermore, the imidazo portion of the tetrahydroimidazopyridine moietyin the structure of formula (I) exists in tautomeric forms, illustratedbelow for a fragment of the compound of Example 1

According to the IUPAC convention, these representations give rise todifferent numbering of the atoms of the imidazole portion:2-(1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(structure A) vs.2-(1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine(structure B). It will be understood that although structures are shown,or named, in a particular form, the invention also includes the tautomerthereof.

The compounds of the invention may contain one or more chiral centersand therefore, such compounds (and intermediates thereof) can exist asracemic mixtures; pure stereoisomers (i.e., enantiomers ordiastereomers); stereoisomer-enriched mixtures and the like. Chiralcompounds shown or named herein without a defined stereochemistry at achiral center are intended to include any or all possible stereoisomervariations at the undefined stereocenter unless otherwise indicated. Thedepiction or naming of a particular stereoisomer means the indicatedstereocenter has the designated stereochemistry with the understandingthat minor amounts of other stereoisomers may also be present unlessotherwise indicated, provided that the utility of the depicted or namedcompound is not eliminated by the presence of another stereoisomer.

Compounds of formula (I) also contain several basic groups (e.g., aminogroups) and therefore, such compounds can exist as the free base or invarious salt forms, such a mono-protonated salt form, a di-protonatedsalt form, a tri-protonated salt form, or mixtures thereof. All suchforms are included within the scope of this invention, unless otherwiseindicated.

This invention also includes isotopically-labeled compounds of formula(I), i.e., compounds of formula (I) where an atom has been replaced orenriched with an atom having the same atomic number but an atomic massdifferent from the atomic mass that predominates in nature. Examples ofisotopes that may be incorporated into a compound of formula (I)include, but are not limited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³⁵S ³⁶Cl, and ¹⁸F. Of particular interest are compounds offormula (I) enriched in tritium or carbon-14, which compounds can beused, for example, in tissue distribution studies.

Also of particular interest are compounds of formula (I) enriched indeuterium especially at a site of metabolism, which compounds areexpected to have greater metabolic stability. Additionally of particularinterest are compounds of formula (I) enriched in a positron emittingisotope, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, which compounds can be used, forexample, in Positron Emission Tomography (PET) studies.

Definitions

When describing this invention including its various aspects andembodiments, the following terms have the following meanings, unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Unless otherwisedefined, such alkyl groups typically contain from 1 to 10 carbon atoms.Representative alkyl groups include, by way of example, methyl (Me),ethyl (Et), n-propyl (n-Pr) or (nPr), isopropyl (i-Pr) or (iPr), n-butyl(n-Bu) or (nBu), sec-butyl, isobutyl, tert-butyl (t-Bu) or (tBu),n-pentyl, n-hexyl, 2,2-dimethylpropyl, 2-methylbutyl, 3-methylbutyl,2-ethylbutyl, 2,2-dimethylpentyl, 2-propylpentyl, and the like.

The term “alkylene” means a divalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Unless otherwisedefined, such alkyl groups typically contain from 1 to 10 carbon atoms.Representative alkylene groups include, by way of example —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, and the like.

When a specific number of carbon atoms are intended for a particularterm, the number of carbon atoms is shown preceding the term. Forexample, the term “C₁₋₃ alkyl” means an alkyl group having from 1 to 3carbon atoms wherein the carbon atoms are in any chemically-acceptableconfiguration, including linear or branched configurations.

The term “cycloalkyl” means a monovalent saturated carbocyclic groupwhich may be monocyclic or multicyclic. Unless otherwise defined, suchcycloalkyl groups typically contain from 3 to 10 carbon atoms.Representative cycloalkyl groups include, by way of example, cyclopropyl(cPr), cyclobutyl (cBu), cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, adamantyl, and the like.

The term “heterocycle”, “heterocyclic”, or “heterocyclic ring” means amonovalent saturated or partially unsaturated cyclic non-aromatic group,having from 3 to 10 total ring atoms, wherein the ring contains from 2to 9 carbon ring atoms and from 1 to 4 ring heteroatoms selected fromnitrogen, oxygen, and sulfur. Heterocyclic groups may be monocyclic ormulticyclic (i.e., fused or bridged). Representative heterocyclic groupsinclude, by way of example, pyrrolidinyl, piperidinyl, piperazinyl,imidazolidinyl, morpholinyl, thiomorpholyl, indolin-3-yl,2-imidazolinyl, tetrahydropyranyl, 1,2,3,4-tetrahydroisoquinolin-2-yl,quinuclidinyl, 7-azanorbornanyl, nortropanyl, and the like, where thepoint of attachment is at any available carbon or nitrogen ring atom.Where the context makes the point of attachment of the heterocyclicgroup evident, such groups may alternatively be referred to as anon-valent species, i.e. pyrrolidine, piperidine, piperazine, imidazole,tetrahydropyran etc.

The term “halo” means fluoro, chloro, bromo or iodo.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a compound of the invention or apharmaceutically-acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent.

Representative solvents include by way of example, water, methanol,ethanol, isopropanol, acetic acid, and the like. When the solvent iswater, the solvate formed is specifically termed a hydrate.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treatment” as used herein means the treatment of a disease,disorder, or medical condition (such as a respiratory disease), in apatient, such as a mammal (particularly a human) which includes one ormore of the following:

(a) preventing the disease, disorder, or medical condition fromoccurring, i.e., preventing the reoccurrence of the disease or medicalcondition or prophylactic treatment of a patient that is pre-disposed tothe disease or medical condition;

(b) ameliorating the disease, disorder, or medical condition, i.e.,eliminating or causing regression of the disease, disorder, or medicalcondition in a patient, including counteracting the effects of othertherapeutic agents;

(c) suppressing the disease, disorder, or medical condition, i.e.,slowing or arresting the development of the disease, disorder, ormedical condition in a patient; or

(d) alleviating the symptoms of the disease, disorder, or medicalcondition in a patient.

The term “pharmaceutically acceptable salt” means a salt that isacceptable for administration to a patient or a mammal, such as a human(e.g., salts having acceptable mammalian safety for a given dosageregime). Representative pharmaceutically acceptable salts include saltsof acetic, ascorbic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, edisylic, fumaric, gentisic, gluconic, glucoronic,glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic,lactobionic, maleic, malic, mandelic, methanesulfonic, mucic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic, nicotinic, nitric, orotic, pamoic,pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonicand xinafoic acid, and the like.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. For example, the cation can be a protonated form ofa compound of formula (I), i.e. a form where one or more amino groupshave been protonated by an acid. Typically, the salt is apharmaceutically acceptable salt, although this is not required forsalts of intermediate compounds that are not intended for administrationto a patient.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino nitrogen. Representativeamino-protecting groups include, but are not limited to, formyl; acylgroups, for example alkanoyl groups, such as acetyl andtri-fluoroacetyl; alkoxycarbonyl groups, such as tert butoxycarbonyl(Boc); arylmethoxycarbonyl groups, such as benzyloxycarbonyl (Cbz) and9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups, such as benzyl(Bn), trityl (Tr), and 1,1-di-(4′-methoxyphenyl)methyl; silyl groups,such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS),[2-(trimethylsilyl)ethoxy]methyl (SEM); and the like. Numerousprotecting groups, and their introduction and removal, are described inT. W. Greene and P. G. M. Wuts, Protecting Groups in Organic Synthesis,Third Edition, Wiley, New York

General Synthetic Procedures

Compounds of this invention, and intermediates thereof, can be preparedaccording to the following general methods and procedures usingcommercially-available or routinely-prepared starting materials andreagents. The substituents and variables (e.g., R¹, R², R³, R⁴, etc.)used in the following schemes have the same meanings as those definedelsewhere herein unless otherwise indicated. Additionally, compoundshaving an acidic or basic atom or functional group may be used or may beproduced as a salt unless otherwise indicated (in some cases, the use ofa salt in a particular reaction will require conversion of the salt to anon-salt form, e.g., a free base, using routine procedures beforeconducting the reaction).

Although a particular embodiment of the present invention may be shownor described in the following procedures, those skilled in the art willrecognize that other embodiments or aspects of the present invention canalso be prepared using such procedures or by using other methods,reagents, and starting materials know to those skilled in the art. Inparticular, it will be appreciated that compounds of the invention maybe prepared by a variety of process routes in which reactants arecombined in different orders to provide different intermediates en routeto producing final products.

A general method of preparing final compounds of the invention utilizesa key intermediate 1 as illustrated in Scheme 1. The variables R², R³,R⁴, and R⁷ are defined as in formula (I), R¹ is hydrogen, Pg representsan amino protecting group, typically Boc, and R^(8a) and R^(8b) aredefined so that the group R⁸ is formed upon completion of the reaction,i.e. R^(8a)—C(H)—R^(8b) is R⁸. For example, when R⁸ is methyl, thevariables R^(8a) and R^(8b) are each hydrogen such thatR^(8a)—C(═O)—R^(8b) is formaldehyde. For R⁸ defined as isopropyl, R^(8a)and R^(8b) are each methyl such that R^(8a)—C(═O)—R^(8b) is acetone.

In Scheme 1, intermediate 1 is reductively N-alkylated by reaction withthe aldehyde or ketone 2 to provide the protected intermediate 3. Thereaction is typically conducted by contacting intermediate 1 withbetween about 1 and about 2 equivalents of compound 2 in a suitableinert diluent, such as dichloromethane, methanol, tetrahydrofuran, ordimethylformamide in the presence of between about 2 and about 4equivalents of a reducing agent. Between about 2 and about 3 equivalentsof acetic acid may optionally be included in the reaction. The reactionis typically conducted at a temperature in the range of about 20° C. toabout 40° C. for about 2 to about 48 hours or until the reaction issubstantially complete. Typical reducing agents include sodiumtriacetoxyborohydride and sodium cyanoborohydride.

The protecting group is removed from intermediate 3 under typicalconditions. For example, a Boc group may be removed by standardtreatment with an acid, typically trifluoroacetic acid or hydrochloricacid in dioxane to provide intermediate 4 which is reacted with acompound of the formula R^(8a)—C(═O)—R^(8b) under similar reductivealkylation conditions as in the first step to provide a final compound(I)′.

For compounds in which the group of formula (II) includes a tertiarynitrogen, for example, where R⁷ and R⁸ taken together form C₃₋₅alkyleneor C₂alkylene-O—C₂alkylene, final compounds may be directly prepared bya reaction of intermediate 1 with an intermediate 2′

in which the amino protecting group Pg of the compound of formula 2 isreplaced by R⁸. For example, as illustrated in Examples 6 and 17 below,final compounds of formula (IV) may be prepared by reaction ofintermediate 1 with a compound of formula 2″:

A useful process for the preparation of intermediate 1 is illustrated inScheme 2.

As described in detail in Preparations 9 and 10, and also 13 and 14,below, the bromoindazole aldehyde 5 is reacted with the benzyl protectedimine compound 6 to provide intermediate 7. The reaction is typicallyconducted in the presence of sodium bisulfite, at a temperature ofbetween about 130° C. and about 140° C. for between about 1 and about 6hours or until the reaction is substantially complete. The product maybe isolated by precipitation from the reaction mixture followed byfreebasing and recrystallization. Compound 7 is reduced using a reducingagent such as sodium borohydride to provide compound 8. The reaction isbeneficially performed in a diluent composed of methyltetrahydrofuran,methanol, and water.

Isolation of the product 8 as a freebase or as a hydrochloride saltprovides a product of excellent purity. Intermediate 8 is combined withprotected phenyltrifluoroborate 9 under typical Suzuki-Miyaura couplingconditions to provide intermediate 10. The reaction is typicallyconducted at elevated temperature in the presence of a palladiumcatalyst. Optionally, the Suzuki coupling reaction is promoted by theinclusion of an additional agent prepared by the reaction ofbis(pinacolato)boron with potassium hydrogen difluoride, as described inPreparation 16. Finally, the benzyl groups of intermediate 10 areremoved under typical conditions, for example in a hydrogen atmospherein the presence of a palladium catalyst, to provide intermediate 1.

The imine compound 6 used in the first step of Scheme 2 is convenientlyprepared by reacting a pyridine diamine with benzyl bromide and issupplied as the hydrobromide salt. As described in Preparation 8, theSuzuki partner 9, shown in Scheme 2 as the trifluoroborate potassiumsalt can be prepared by benzyl protecting 4-bromo-5-ethyl-2-fluorophenolby reaction with benzyl bromide and reacting the benzyl protected phenolwith bis(pinacolato)diboron to prepare the boronate which issubsequently reacted with potassium hydrogen difluoride to provideintermediate 9. Alternatively, a boronate intermediate can be used inplace of the trifluoroborate 9.

Accordingly, in a method aspect, the invention provides a process ofpreparing a compound of formula (I′) or a pharmaceutically acceptablesalt thereof, the process comprising (a) reacting a compound of formula1 with a compound of formula 2 to provide an intermediate of formula 3,(b) deprotecting intermediate 3 to provide intermediate 4, and (c)reacting intermediate 4 with R^(8a)—C(═O)—R^(8b) to provide a compoundof formula (I′) or a pharmaceutically acceptable salt thereof. Theinvention further provides a process of preparing a compound of formula(IV) or a pharmaceutically-acceptable salt thereof, the processcomprising reacting a compound of formula 1 with a compound of formula2″ to provide a compound of formula (IV) or a pharmaceuticallyacceptable salt thereof.

In a further method aspect, the invention provides a process ofpreparing a compound of formula 1, the process comprising (a) reacting acompound of formula 8 with a compound of formula 9 to provide a compoundof formula 10, and (b) deprotecting the compound of formula 10 toprovide a compound of formula 1.

In yet another aspect, the invention provides a compound of formula 8and a hydrochloride salt thereof useful in the preparation ofintermediate 1.

Crystalline Form

In another aspect, the invention provides the crystalline hydrate of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

The crystalline hydrate of the invention is a crystalline freebase ofthe compound of Example 6. In one aspect, the crystalline hydrate ischaracterized by a powder X-ray diffraction (PXRD) pattern havingsignificant diffraction peaks, among other peaks, at 20 values of6.20±0.20, 9.58±0.20, 17.53±0.20, 19.28±0.20, and 21.51±0.20. Thecrystalline hydrate may be further characterized by a PXRD patternhaving two or more additional diffraction peaks, including three or moreand four or more additional diffraction peaks at 2θ values selected from10.34±0.20, 11.54±0.20, 12.77±0.20, 13.01±0.20, 16.94±0.20, 20.61±0.20,and 22.10±0.20. In another aspect, the crystalline hydrate ischaracterized by a PXRD pattern having diffraction peaks at 2θ values of6.20±0.20, 9.58±0.20, 10.34±0.20, 11.54±0.20, 12.77±0.20, 13.01±0.20,16.94±0.20, 17.53±0.20, 19.28±0.20, 20.61±0.20, 21.51±0.20, and22.10±0.20.

As is well known in the field of powder X-ray diffraction, peakpositions of PXRD spectra are relatively less sensitive to experimentaldetails, such as details of sample preparation and instrument geometry,than are the relative peak heights. Thus, in one aspect, the crystallinehydrate is characterized by a powder x-ray diffraction pattern in whichthe peak positions are substantially in accordance with those shown inFIG. 1.

In another aspect, the crystalline hydrate is characterized by itsbehavior when exposed to high temperature. As demonstrated in FIG. 2,the differential scanning calorimetry (DSC) trace recorded at a heatingrate of 10° C. per minute exhibits a desolvation endotherm with an onsetat about 83° C. and a peak at about 128° C. and a peak in endothermicheat flow, identified as a melt transition, in the range of about 206°C. to about 216° C., including between about 209° C. and about 214° C.The thermal gravimetric analysis (TGA) trace of FIG. 3 shows adesolvation onset at a temperature of about 112° C. and a decompositiononset at a temperature of about 250° C. The TGA profile shows a weightloss of about 3.86% at 190° C. which may be interpreted as the loss ofwater and compared with the theoretical weight percentage of water for amonohydrate of 3.65%. Accordingly it is believed the present crystallinehydrate is a monohydrate.

The present crystalline hydrate has been demonstrated to have areversible sorption/desorption profile with an exceptionally smallpropensity for hygroscopicity. Form I demonstrated less than about 0.12%weight gain in the humidity range of 5% to 90% relative humidity asshown in FIG. 4. No hysteresis was observed in two cycles of sorptionand desorption. The crystalline hydrate is considered to benon-hygroscopic.

The crystalline hydrate of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolis conveniently prepared by a slurry form conversion of a slurry of thereaction product of the reductive N-alkylation reaction of theintermediate of formula 1 with 1-methylpiperidin-4-one. After an initialquench of the reductive N-alkylation reaction with ammonia in water, theresulting slurry is diluted in a protic solvent, for example methanol,ethanol, isopropyl alcohol, or n-propyl alcohol, and heated at atemperature of from about 40° C. to about 60° C. for between about 1 andabout 24 hours or until conversion to a solvate form is complete. Whilehot, water is added as an antisolvent to precipitate a solvate of thereaction product, which is cooled, for example, to about 10° C. Theprecipitate is washed with a 1:1 mixture of water with the proticsolvent. Typically, the solvate includes the diluent in which thereductive alkylation reaction was performed, the protic solvent, andwater.

The slurry form conversion to the crystalline hydrate of the inventionis performed by forming a slurry of either the solvate formed asdescribed above or of amorphous5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolin a diluent including from about 1 to about 30% v/v water along with anorganic solvent. Useful organic solvents for the form conversioninclude, but are not limited to methanol, tetrahydrofuran, tert-butylalcohol, acetonitrile, isopropylacetate and acetone. The form conversionoptionally includes heating, for example heating at from about 40° C. toabout 60° C. for between about 1 hour and about 2 days or until the formconversion is complete. As described in Example 17, methanol is usefulas the protic solvent in the initial step while acetone is particularlyuseful for the slurry form conversion.

Accordingly in a method aspect, the invention provides a method ofpreparing the crystalline hydrate of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,the method comprising (a) forming a slurry of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolin solvate or amorphous form in a diluent including from about 1 toabout 30% v/v water along with an organic solvent selected frommethanol, tetrahydrofuran, tert-butyl alcohol, acetonitrile,isopropylacetate and acetone, (b) heating the slurry at a temperaturebetween about 40° C. and about 60° C. for between about 1 hour and about2 days, and (c) isolating the crystalline hydrate from the slurry.

Pharmaceutical Compositions

The compounds of the invention and pharmaceutically-acceptable saltsthereof are typically used in the form of a pharmaceutical compositionor formulation. Such pharmaceutical compositions may advantageously beadministered to a patient by inhalation. In addition, pharmaceuticalcompositions may be administered by any acceptable route ofadministration including, but not limited to, oral, rectal, nasal,topical (including transdermal) and parenteral modes of administration.

Accordingly, in one of its compositions aspects, the invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a compound offormula (I), where, as defined above, “compound of formula (I)” means acompound of formula (I) or a pharmaceutically-acceptable salt thereof.Optionally, such pharmaceutical compositions may contain othertherapeutic and/or formulating agents if desired. When discussingcompositions and uses thereof, the “compound of the invention” may alsobe referred to herein as the “active agent”. As used herein, the term“compound of the invention” is intended to include all compoundsencompassed by formula (I) as well as the species embodied in formulas(III), (IV), and (V) and pharmaceutically-acceptable salts thereof

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the present invention.Those skilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,i.e., bulk compositions, or less than a therapeutically effectiveamount, i.e., individual unit doses designed for multiple administrationto achieve a therapeutically effective amount.

Typically, such pharmaceutical compositions will contain from about 0.01to about 95% by weight of the active agent; including, for example, fromabout 0.05 to about 30% by weight; and from about 0.1% to about 10% byweight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the carriers or excipients used inthe pharmaceutical compositions of this invention arecommercially-available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20th Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7th Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with apharmaceutically-acceptable carrier and one or more optionalingredients. The resulting uniformly blended mixture can then be shapedor loaded into tablets, capsules, pills and the like using conventionalprocedures and equipment.

In one aspect, the pharmaceutical composition is suitable for inhaledadministration. Pharmaceutical compositions for inhaled administrationare typically in the form of an aerosol or a powder. Such compositionsare generally administered using inhaler delivery devices, such as a drypowder inhaler (DPI), a metered-dose inhaler (MDI), a nebulizer inhaler,or a similar delivery device.

In a particular embodiment, the pharmaceutical composition isadministered by inhalation using a dry powder inhaler. Such dry powderinhalers typically administer the pharmaceutical composition as afree-flowing powder that is dispersed in a patient's air-stream duringinspiration. In order to achieve a free-flowing powder composition, thetherapeutic agent is typically formulated with a suitable excipient suchas lactose, starch, mannitol, dextrose, polylactic acid (PLA),polylactide-co-glycolide (PLGA) or combinations thereof. Typically, thetherapeutic agent is micronized and combined with a suitable carrier toform a composition suitable for inhalation.

A representative pharmaceutical composition for use in a dry powderinhaler comprises lactose and a compound of the invention in micronizedform. Such a dry powder composition can be made, for example, bycombining dry milled lactose with the therapeutic agent and then dryblending the components. The composition is then typically loaded into adry powder dispenser, or into inhalation cartridges or capsules for usewith a dry powder delivery device.

Dry powder inhaler delivery devices suitable for administeringtherapeutic agents by inhalation are described in the art and examplesof such devices are commercially available. For example, representativedry powder inhaler delivery devices or products include Aeolizer(Novartis); Airmax (IVAX); ClickHaler (Innovata Biomed); Diskhaler(GlaxoSmithKline); Diskus/Accuhaler (GlaxoSmithKline); Ellipta(GlaxoSmithKline); Easyhaler (Orion Pharma); Eclipse (Aventis); FlowCaps(Hovione); Handihaler (Boehringer Ingelheim); Pulvinal (Chiesi);Rotahaler (GlaxoSmithKline); SkyeHaler/Certihaler (SkyePharma);Twisthaler (Schering-Plough); Turbuhaler (AstraZeneca); Ultrahaler(Aventis); and the like.

In another particular embodiment, the pharmaceutical composition isadministered by inhalation using a metered-dose inhaler. Suchmetered-dose inhalers typically discharge a measured amount of atherapeutic agent using a compressed propellant gas. Accordingly,pharmaceutical compositions administered using a metered-dose inhalertypically comprise a solution or suspension of the therapeutic agent ina liquefied propellant. Any suitable liquefied propellant may beemployed including hydrofluoroalkanes (HFAs), such as1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227); and chlorofluorocarbons,such as CCl₃F. In a particular embodiment, the propellant ishydrofluoroalkanes. In some embodiments, the hydrofluoroalkaneformulation contains a co-solvent, such as ethanol or pentane, and/or asurfactant, such as sorbitan trioleate, oleic acid, lecithin, andglycerin.

A representative pharmaceutical composition for use in a metered-doseinhaler comprises from about 0.010% to about 5% by weight of a compoundof the invention; from about 0% to about 20% by weight ethanol; and fromabout 0% to about 5% by weight surfactant; with the remainder being anHFA propellant. Such compositions are typically prepared by addingchilled or pressurized hydrofluoroalkane to a suitable containercontaining the therapeutic agent, ethanol (if present) and thesurfactant (if present). To prepare a suspension, the therapeutic agentis micronized and then combined with the propellant. The composition isthen loaded into an aerosol canister, which typically forms a portion ofa metered-dose inhaler device.

Metered-dose inhaler devices suitable for administering therapeuticagents by inhalation are described in the art and examples of suchdevices are commercially available. For example, representativemetered-dose inhaler devices or products include AeroBid Inhaler System(Forest Pharmaceuticals); Atrovent Inhalation Aerosol (BoehringerIngelheim); Flovent (GlaxoSmithKline); Maxair Inhaler (3M); ProventilInhaler (Schering); Serevent Inhalation Aerosol (GlaxoSmithKline); andthe like.

In another particular aspect, the pharmaceutical composition isadministered by inhalation using a nebulizer inhaler. Such nebulizerdevices typically produce a stream of high velocity air that causes thepharmaceutical composition to spray as a mist that is carried into thepatient's respiratory tract. Accordingly, when formulated for use in anebulizer inhaler, the therapeutic agent can be dissolved in a suitablecarrier to form a solution. Alternatively, the therapeutic agent can bemicronized or nanomilled and combined with a suitable carrier to form asuspension.

A representative pharmaceutical composition for use in a nebulizerinhaler comprises a solution or suspension comprising from about 0.05μg/mL to about 20 mg/mL of a compound of the invention and excipientscompatible with nebulized formulations. In one embodiment, the solutionhas a pH of about 3 to about 8.

Nebulizer devices suitable for administering therapeutic agents byinhalation are described in the art and examples of such devices arecommercially available. For example, representative nebulizer devices orproducts include the Respimat Softmist Inhalaler (Boehringer Ingelheim);the AERx Pulmonary Delivery System (Aradigm Corp.); the PARI LC PlusReusable Nebulizer (Pari GmbH); and the like.

In yet another aspect, the pharmaceutical compositions of the inventionmay alternatively be prepared in a dosage form intended for oraladministration. Suitable pharmaceutical compositions for oraladministration may be in the form of capsules, tablets, pills, lozenges,cachets, dragees, powders, granules; or as a solution or a suspension inan aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oilliquid emulsion; or as an elixir or syrup; and the like; each containinga predetermined amount of a compound of the present invention as anactive ingredient.

When intended for oral administration in a solid dosage form, thepharmaceutical compositions of the invention will typically comprise theactive agent and one or more pharmaceutically-acceptable carriers, suchas sodium citrate or dicalcium phosphate. Optionally or alternatively,such solid dosage forms may also comprise: fillers or extenders,binders, humectants, solution retarding agents, absorption accelerators,wetting agents, absorbents, lubricants, coloring agents, and bufferingagents. Release agents, wetting agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the pharmaceutical compositions of the invention.

Alternative formulations may also include controlled releaseformulations, liquid dosage forms for oral administration, transdermalpatches, and parenteral formulations. Conventional excipients andmethods of preparation of such alternative formulations are described,for example, in the reference by Remington, supra.

The following non-limiting examples illustrate representativepharmaceutical compositions of the present invention.

Dry Powder Composition

A micronized compound of formula (I) (1 g) is blended with milledlactose (25 g). This blended mixture is then loaded into individualblisters of a peelable blister pack in an amount sufficient to providebetween about 0.1 mg to about 4 mg of the compound of formula I perdose. The contents of the blisters are administered using a dry powderinhaler.

Dry Powder Composition

A micronized compound of formula (I) (1 g) is blended with milledlactose (20 g) to form a bulk composition having a weight ratio ofcompound to milled lactose of 1:20. The blended composition is packedinto a dry powder inhalation device capable of delivering between about0.1 mg to about 4 mg of the compound of formula I per dose.

Metered-Dose Inhaler Composition

A micronized compound of formula (I) (10 g) is dispersed in a solutionprepared by dissolving lecithin (0.2 g) in demineralized water (200 mL).The resulting suspension is spray dried and then micronized to form amicronized composition comprising particles having a mean diameter lessthan about 1.5 μm. The micronized composition is then loaded intometered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 0.1mg to about 4 mg of the compound of formula I per dose when administeredby the metered dose inhaler.

Nebulizer Composition

A compound of formula (I) (25 mg) is dissolved in a solution containing1.5-2.5 equivalents of hydrochloric acid, followed by addition of sodiumhydroxide to adjust the pH to 3.5 to 5.5 and 3% by weight of glycerol.The solution is stirred well until all the components are dissolved. Thesolution is administered using a nebulizer device that provides about0.1 mg to about 4 mg of the compound of formula I per dose.

Utility

The JAK inhibitors of the invention have been designed for the treatmentof inflammatory and fibrotic disease of the respiratory tract. Inparticular, the compounds have been designed to enable delivery of apotent anti-cytokine agent directly to the site of action of respiratorydisease in the lung while limiting systemic exposure.

The compounds of the invention have been shown to be potent inhibitorsof the JAK family of enzymes: JAK1, JAK2, JAK3, and TYK2. In addition,the compounds have demonstrated potent inhibition of pro-inflammatoryand pro-fibrotic cytokines without exhibiting cytotoxicity in cellularassays. It has been recognized that the broad anti-inflammatory effectof JAK inhibitors could suppress normal immune cell function,potentially leading to increased risk of infection. The presentcompounds have therefore been optimized to limit absorption from thelung into the plasma, thus minimizing the risk of immunosuppression.

As described in the experimental section below, the absorption anddistribution of typical compounds has been profiled in preclinicalassays. Selected compounds tested in mice showed, at the same time, highconcentration in lung tissue and low absorption into plasma. Compoundstested in mouse exhibited exposure in lung from one to two orders ofmagnitude greater than exposure in plasma. The compounds also exhibitedsignificant retention in the mouse lung as evidenced by a lung half-lifegreater than about 5 hours. Importantly, the concentration of testcompound in the mouse lung has been shown to correlate with a predictedpharmacodynamic effect of JAK enzyme inhibition. Compounds of theinvention have been shown to inhibit an effect of the pro-inflammatorycytokine IL-13 in mouse lung tissue. Specifically, the compounds havedemonstrated dose and concentration dependent inhibition ofIL-13-induced phosphorylation of STAT6 in lung tissue which providesevidence of local lung JAK target engagement in vivo. This effect hasbeen observed when the pro-inflammatory cytokine IL-13 is administered 4hours after administration of the test compound, providing furtherevidence of significant retention in the lung.

Tested compounds have been demonstrated to exhibit both potentinhibitory activity at the cellular level and significant retention inlung tissue. Extensive investigation by the present inventors hasdetermined that while it is possible to identify compounds that arepotent at the cellular level or compounds that show significantretention in the lung, it is far more difficult to discover compoundsthat exhibit both desirable characteristics at the same time.

The diamino structure of the compounds of the present invention,including two amino nitrogen atoms, has been shown to be critical insatisfying both criteria of cellular potency and lung retention. Asdescribed in the assay section below, a compound in which the nitrogenatom in the group of formula (II) is replaced by a carbon atom does notsatisfy both criteria. Not only is such a monoamino compound noticeablyless potent at the cellular level than the corresponding diaminocompound, but it does not exhibit significant inhibition in thepharmacodynamic assay nor exhibit high concentration in lung tissueunder the same assay conditions in which the present compounds showsignificant retention in the lung.

Further, compounds of the invention have been demonstrated to showsufficient solubility at pH values compatible with formulations foradministration by nebulization. Solubility may also be relevant totoxicity testing of compounds intended to be administered by inhalation.It has been observed that administration of undissolved particulatematter by inhalation can be associated with adverse lung effects duringtoxicity testing (Jones et al, Xenobiotica, 2011, 1-8). Solubility ofthe present compounds may also facilitate assessment of toxicity byinhalation.

The anti-inflammatory activity of JAK inhibitors has been robustlydemonstrated in preclinical models of asthma (Malaviya et al., IntImmunopharmacol, 2010, 10, 829-836; Matsunaga et al., Biochem andBiophys Res Commun, 2011, 404, 261-267; Kudlacz et al., Eur J Pharmacol,2008, 582, 154-161.) Accordingly, the compounds of the invention areexpected to be useful for the treatment of inflammatory respiratorydisorders, in particular, asthma. Inflammation and fibrosis of the lungis characteristic of other respiratory diseases in addition to asthmasuch as chronic obstructive pulmonary disease (COPD), cystic fibrosis(CF), pneumonitis, interstitial lung diseases (including idiopathicpulmonary fibrosis), acute lung injury, acute respiratory distresssyndrome, bronchitis, emphysema, and bronchiolitis obliterans. Thepresent compounds, therefore, are also expected to be useful for thetreatment of chronic obstructive pulmonary disease, cystic fibrosis,pneumonitis, interstitial lung diseases (including idiopathic pulmonaryfibrosis), acute lung injury, acute respiratory distress syndrome,bronchitis, emphysema and bronchiolitis obliterans.

In one aspect, therefore, the invention provides a method of treating arespiratory disease in a mammal (e.g., a human), the method comprisingadministering to the mammal a therapeutically-effective amount of acompound of the invention or of a pharmaceutical composition comprisinga pharmaceutically-acceptable carrier and a compound of the invention.

In one aspect, the respiratory disease is asthma, chronic obstructivepulmonary disease, cystic fibrosis, pneumonitis, chronic obstructivepulmonary disease (COPD), cystic fibrosis (CF), pneumonitis,interstitial lung diseases (including idiopathic pulmonary fibrosis),acute lung injury, acute respiratory distress syndrome, bronchitis,emphysema or bronchiolitis obliterans. In another aspect, therespiratory disease is asthma or chronic obstructive pulmonary disease.

The invention further provides a method of treating asthma in a mammal,the method comprising administering to the mammal atherapeutically-effective amount of a compound of the invention or of apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a compound of the invention.

When used to treat asthma, the compounds of the invention will typicallybe administered in a single daily dose or in multiple doses per day,although other forms of administration may be used. The amount of activeagent administered per dose or the total amount administered per daywill typically be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered and itsrelative activity, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.

Compounds of the invention have been demonstrated to be potentinhibitors of the JAK1, JAK2, JAK3, and TYK2 enzymes in enzyme bindingassays, to have potent functional activity without cytotoxicity incellular assays, and to exert the pharmacodynamic effects of JAKinhibition in preclinical models, as described in the followingexamples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention.

In the examples below, the following abbreviations have the followingmeanings unless otherwise indicated. Abbreviations not defined belowhave their generally accepted meanings.

ACN=acetonitrile

CPME=cyclopentyl methyl ether

DCM=dichloromethane

DIPEA=N,N-diisopropylethylamine

DMAc=dimethylacetamide

DMF=N,N-dimethylformamide

EtOAc=ethyl acetate

h=hour(s)

IPAc=isopropylacetate

KOAc=potassium acetate

MeOH=methanol

MeTHF=2-methyltetrahydrofuran

min=minute(s)

MTBE=methyl tert-butyl ether

NMP=N-methyl-2-pyrrolidone

Pd(amphos)₂Cl₂=bis(di-tert-butyl(4-dimethylaminophenyl)-phosphine)dichloropalladium(II)

Pd(dppf)Cl₂=dichloro(1,1′-bis(diphenylphosphino)-ferrocene)dipalladium(II)

Pd(PPh₃)₄=tetrakis(triphenylphosphine)palladium(0)

Pd(t-Bu₃P)₂=bis(tri-tert-butylphosphine) palladium(0)

RT=room temperature

TEA=triethylamine

TFA=trifluoroacetic acid

THF=tetrahydrofuran

bis(pinacolato)diboron=4,4,5,5,4′,4′,5′,5′-octamethyl-[2,2′]bi[[1,3,2]dioxaborolanyl]

Reagents and solvents were purchased from commercial suppliers (Aldrich,Fluka, Sigma, etc.), and used without further purification. Progress ofreaction mixtures was monitored by thin layer chromatography (TLC),analytical high performance liquid chromatography (anal. HPLC), and massspectrometry. Reaction mixtures were worked up as described specificallyin each reaction; commonly they were purified by extraction and otherpurification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by column chromatography or by preparative HPLC,typically using C18 or BDS column packings and conventional eluents.Typical preparative HPLC conditions are described below.

Characterization of reaction products was routinely carried out by massand ¹H-NMR spectrometry. For NMR analysis, samples were dissolved indeuterated solvent (such as CD₃OD, CDCl₃, or d₆-DMSO), and ¹H-NMRspectra were acquired with a Varian Gemini 2000 instrument (400 MHz)under standard observation conditions. Mass spectrometric identificationof compounds was performed by an electrospray ionization method (ESMS)with an Applied Biosystems (Foster City, Calif.) model API 150 EXinstrument or a Waters (Milford, Mass.) 3100 instrument, coupled toautopurification systems.

Preparative HPLC Conditions Column: C18, 5 μm. 21.2×150 mm or C18, 5 μm21×250 or

-   -   C14, 5 μm 21×150 mm        Column temperature: Room Temperature        Flow rate: 20.0 mL/min

Mobile Phases: A=Water+0.05% TFA

-   -   B=ACN+0.05% TFA,        Injection volume: (100-1500 μL)        Detector wavelength: 214 nm

Crude compounds were dissolved in 1:1 water:acetic acid at about 50mg/mL. A 4 minute analytical scale test run was carried out using a2.1×50 mm C18 column followed by a 15 or 20 minute preparative scale runusing 100 μL injection with the gradient based on the % B retention ofthe analytical scale test run. Exact gradients were sample dependent.Samples with close running impurities were checked with a 21×250 mm C18column and/or a 21×150 mm C14 column for best separation. Fractionscontaining desired product were identified by mass spectrometricanalysis.

Analytic HPLC Conditions Method A

Column: Advanced Material Technology HALO® C18 (2), 150×4.60 nm, 2.7micronColumn temperature: 30° C.Flow rate: 1.0 mL/minInjection volume: 5 μLSample preparation: Dissolve in 1:1 ACN:water

Mobile Phases: A=Water:ACN:TFA (98:2:0.1)

-   -   B=Water:ACN:TFA (30:70:0.1)        Detector wavelength: 254 nm        Gradient: 22 min total (time (min)/% B): 0/30, 15/100, 18/100,        20/30, 22/30

Method B

Column: Agilent Zorbax Bonus-RP C18, 150×4.60 nm, 3.5 micronColumn temperature: 40° C.Flow rate: 1.5 mL/minInjection volume: 5 μLSample preparation: Dissolve in 1:1 ACN:1 M HCl

Mobile Phases: A=Water:TFA (99.95:0.05)

-   -   B=ACN:TFA (99.95:0.05)        Detector wavelength: 254 nm and 214 nm        Gradient: 26 min total (time (min)/% B): 0/5, 18/90, 22/90,        22.5/90, 26/5

Method C Column: Agilent Poroshell 120 Bonus-RP, 4.6×150 mm, 2.7 μm

Column temperature: 30° C.Flow rate: 1.5 mL/minInjection volume: 10 μL

Mobile Phases: A=ACN:Water:TFA (2:98:0.1)

-   -   B=ACN:Water:TFA (90:10:0.1)        Sample preparation: Dissolve in Mobile phase B        Detector wavelength: 254 nm and 214 nm        Gradient: 60 min total (time (min)/% B): 0/0, 50/100, 55/100,        55.1/0, 60/0

Preparation 1: 1-(Benzyloxy)-4-bromo-5-ethyl-2-fluorobenzene

(a) 5-Ethyl-2-fluorophenol

A mixture of compound 5-bromo-2-fluorophenol (80 g, 419 mmol) in drytetrahydrofuran (800 mL) was degassed and purged with nitrogen 3 times,and Pd(t-Bu₃P)₂ (4.28 g, 8.38 mmol) was added. Diethylzinc (114 g, 921mmol) was added to the mixture dropwise at 25° C., and the reactionmixture was stirred at 50° C. for 12 h under nitrogen and slowly pouredinto ice-water (1 L). EtOAc (350 mL) was added and the reaction mixturewas stirred for 20 min and filtered. The filter cake was washed withEtOAc (3×500 mL). The combined organic layers were washed with brine(600 mL), dried over sodium sulfate, concentrated, and purified bysilica gel chromatography to give the title intermediate (85 g, crude)as a yellow oil.

(b) 2-(Benzyloxy)-4-ethyl-1-fluorobenzene

To a solution of the product of the previous step (85 g, 606 mmol) inACN (850 mL) was added benzyl bromide (124 g, 728 mmol) and K₂CO₃ (126g, 909 mmol). The reaction mixture was stirred at 25° C. for 12 h,poured into water (1 L) and extracted with EtOAc (4×500 mL). Thecombined organic layers were washed with brine (600 mL), dried oversodium sulfate, concentrated, and purified by silica gel chromatographyto give the title intermediate (100 g) as a yellow oil.

(c) 1-(Benzyloxy)-4-bromo-5-ethyl-2-fluorobenzene

To a solution of the product of the previous step (100 g, 434 mmol) inACN (1.0 L) was added N-bromosuccinimide (85 g, 477 mmol) portion wise.The reaction mixture was stirred at 25° C. for 5 h, poured into water(1.3 L) and extracted with EtOAc (3×500 mL). The combined organic layerswere washed with brine (800 mL), dried over sodium sulfate,concentrated, and purified by silica gel chromatography to give thetitle compound (83 g) as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ (ppm)7.27-7.43 (m, 6H), 6.86 (d, J=8.4 Hz, 1H), 5.10 (s, 2H), 2.64 (q, J=7.6Hz, 2H), 1.15 (t, J=7.2 Hz, 1H).

Preparation 2:2-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

A mixture of the compound of Preparation 1 (83 g, 268 mmol),bis(pinacolato)diboron (102 g, 402 mmol), and KOAc (79.0 g, 805 mmol) indioxane (830 mL) was degassed and purged with nitrogen 3 times, andPd(dppf)Cl₂ (3.93 g, 5.37 mmol) was added. The reaction mixture wasstirred at 120° C. for 4 h under nitrogen. The mixture was cooled to 25°C., poured into water (1 L), and extracted with EtOAc (3×500 mL). Thecombined organic layers were washed with brine (800 mL), dried oversodium sulfate, and purified by silica gel chromatography. The productwas washed with methanol (200 mL), filtered, and the filter cake wasdried to give the title compound (65 g) as white solid. ¹H NMR (CDCl₃,400 MHz) δ (ppm) 7.26-7.42 (m, 5H), 6.74 (d, J=7.6 Hz, 1H), 5.08 (s,2H), 2.76 (q, J=7.2 Hz, 2H), 1.25 (s, 12H), 1.06 (t, J=7.6 Hz, 3H).

Preparation 3: 1-Benzyl-4-imino-1,4-dihydropyridin-3-amine

To a solution of pyridine-3,4-diamine (200 g, 1.8 mol) in ACN (17.0 L)was added benzyl bromide (306 g, 1.79 mol) and the reaction mixture wasstirred at 15° C. for 12 h, filtered and the filter cake was dried undervacuum to give the title compound (250 g) as a white solid. ¹H NMR(d₆-DMSO, 400 MHz) δ (ppm) 8.02 (dd, J=7.2, 1.6 Hz, 1H), 7.66 (s, 1H),7.34-7.41 (m, 5H), 6.79 (d, J=6.8 Hz, 1H), 5.62 (s, 2H), 5.36 (s, 2H).

Preparation 4:5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine

(a) 6-Bromo-1H-indazol-3-yl-carbaldehyde

A solution of NaNO₂ (704 g, 10.2 mol) in water (1 L) was added dropwiseto a solution of 6-bromo-1H-indole (400 g, 2.0 mol) in acetone (7 L) at10° C. The reaction mixture was stirred at 10° C. for 30 min, aqueous 3MHCl (437 mL) was added slowly with vigorous stirring, keeping theinternal temperature between 10 and 25° C. The solution was stirred at20° C. for 3 h, and concentrated while keeping the temperature below 35°C. The solid was collected by filtration. The filter cake was washedwith 1:2 petroleum ether:MTBE (800 mL). The solids were collected byfiltration and dried under vacuum to afford the title intermediate (450g) as a black brown solid. ¹H NMR (CH₃OD, 400 MHz) δ (ppm) 7.77 (d,J=8.8 Hz, 1H), 7.69 (s, 1H), 7.22 (dd, J=8.4, 2.4 Hz, 1H), 5.70 (s, 1H).

b) 5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine

To a stirred solution of 6-bromo-1H-indazol-3-yl-carbaldehyde (150.0 g,666 mmol) and 1-benzyl-4-imino-1,4-dihydropyridin-3-amine (127.5 g,639.9 mmol) in DMF (750 mL) was charged NaHSO₃ (83.2 g, 799.9 mmol) andthe reaction mixture was stirred for 6 h at 140° C. and poured intowater (3.5 L). The precipitate was filtered and washed with water (1 L)to give the title compound (180 g) as a black brown solid. ¹H NMR(d₆-DMSO, 400 MHz) δ (ppm) 8.69 (s, 1H) 8.71 (d, J=7.2 Hz, 1H) 8.37 (d,J=8.4 Hz, 1H) 8.07 (d, J=6.4 Hz, 1H) 7.97 (s, 1H) 7.38-7.43 (m, 3H)7.50-7.54 (m, 4H) 5.87 (s, 2H).

Preparation 5:5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

To a solution of5-benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine (23.0 g,56.9 mmol) in MeOH (200 mL) and THF (1 L) was added NaBH₄ (12.9 g, 341.3mmol) portion-wise and the reaction mixture was stirred at 50° C. for 2h. Acetic acid (10 eq) was added, the solution was concentrated todryness and purified by silica gel chromatography (30 g silica, 0-10%MeOH/DCM with 0.1% TEA) to give the title compound (6.0 g). ¹H NMR(d₆-DMSO, 400 MHz) δ (ppm) 8.24 (d, J=8.0 Hz, 1H), 7.77 (s, 1H),7.28-7.37 (m, 7H), 3.74 (s, 2H), 3.48 (br.s, 2H), 2.80 (s, 2H), 2.66 (s,2H).

Preparation 6:5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

(a) tert-Butyl5-benzyl-2-(6-bromo-1-(tert-butoxycarbonyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-1-carboxylate

Two reactions were carried out in parallel. A suspension of5-benzyl-2-(6-bromo-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(80 g, 196 mmol), di-tert-butyl dicarbonate (128 g, 587.8 mmol, 135 mL)and TEA (79.3 g, 784 mmol, 109 mL) in DCM (1 L) was stirred at 20° C.for 12 h. The two reaction suspensions were combined, concentrated todryness, and purified by silica gel chromatography (petroleumether:EtOAc 10:1-0:1) to give the title intermediate (170.0 g).

(b)5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

Two reactions were carried out in parallel. A solution of the product ofthe previous step (85 g, 140 mmol) and 4M HCl in MeOH (400 mL) in DCM(400 mL) was stirred at 25° C. for 12 h. The reaction mixtures werecombined and concentrated to dryness, DCM (250 mL) was added withstirring, and the reaction mixture was stirred for 30 min and filtered.The filter cake was washed with DCM (2×20 mL) and dried to give thetitle compound (85 g) as an off-white solid.

(c)5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

Eighty-five reactions were carried out in parallel. The product of theprevious step (1.0 g, 2.5 mmol),2-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(873 mg, 2.5 mmol), and Pd(PPh₃)₄ (227 mg, 196. μmol) were dissolved ina mixture of water (4 mL) and dioxane (10 mL). The reaction vial wasbubbled with nitrogen for 2 min and Na₂CO₃ (779 mg, 7.4 mmol) was addedquickly under nitrogen. The reaction mixture was heated at 130° C. for1.5 h. The 85 reaction mixtures were combined and concentrated underreduced pressure. The residue was dissolved in DCM (500 mL) and purifiedby silica gel chromatography (150 g silica, eluted with DCM:THF (6:1 to3:1)) to give compound the title compound (50 g) as an off-white solid.

Preparation 7:5-Ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

A mixture of5-benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(44.5 g, 79.8 mmol), Pd(OH)2/C (25 g, 2.7 mmol, 50% purity) and TFA(44.5 g, 390 mmol, 28.9 mL) in MeOH (500 mL) was stirred under hydrogen(50 Psi) for 4 h and filtered. Pd(OH)₂/C (25 g, 2.7 mmol, 50% purity)was added to the filtrate and the resulting suspension was stirred underhydrogen (50 Psi) at 25° C. for 12 h. The suspension was combined withthe suspension from a prior reaction at the 5.5 g scale and filtered.The filter cake was washed with 20:1 MeOH:TFA (2×200 mL). The combinedfiltrate was concentrated and 4 M HCl in MeOH (200 mL) was added to theresidue with stirring. The resulting suspension was concentrated,slurried with MeOH (80 mL) and stirred for 30 min. A white solidprecipitated. The solid was filtered, the filter cake was washed withMeOH (2×10 mL) and dried under vacuum to give the HCl salt of the titlecompound (24.8 g) as an off-white solid. (m/z): [M+H]⁺ calcd forC₂₁H₂₀FN₅O 378.17 found 378.1. ¹H NMR (d₆-DMSO, 400 MHz) δ (ppm) 8.23(d, J=8.4 Hz, 1H), 7.59 (s, 1H), 7.35 (d, J=11.2 Hz, 1H), 6.90-6.97 (m,2H), 4.57 (s, 2H), 3.72 (t, J=6.0 Hz, 2H), 3.22 (t, J=6.0 Hz, 2H), 2.51(q, J=7.6 Hz, 2H), 1.04 (t, J=7.6 Hz, 3H).

Preparation 8: (4-(Benzyloxy)-2-ethyl-5-fluorophenyl)trifluoroborate,potassium

(a) 1-(Benzyloxy)-4-bromo-5-ethyl-2-fluorobenzene

To a mixture of 4-bromo-5-ethyl-2-fluorophenol (50 g, 228 mmol) and DMF(200 mL) was added potassium carbonate (34.7 g, 251 mmol) at RT. Thereaction mixture was stirred for 15 min; benzyl bromide (25.8 mL, 217mmol) was added dropwise; the reaction mixture was stirred at RTovernight and poured into water (1 L). Ethyl acetate (1 L) was added;the phases were separated; the organic layer was washed with brine (1L), and dried with sodium sulfate followed by solvent removal to providethe crude title intermediate (71 g) thick oil. HPLC Method A Retentiontime 17.37 min.

(b)2-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

A mixture of the product of the previous step (70 g, 226 mmol) anddioxane (800 mL) was purged with nitrogen and thenbis(pinacolato)diboron (86 g, 340 mmol) was added followed by potassiumacetate (66.7 g, 679 mmol). The reaction mixture was purged withnitrogen; Pd(dppf)Cl₂ (3.31 g, 4.53 mmol) was added; the reactionmixture was heated at 120° C. under nitrogen for 4 h; cooled to RT andstirred overnight. The reaction mixture was concentrated by rotaryevaporation and partitioned between water (800 mL) and ethyl acetate(800 mL). The organic layer was washed with brine (800 mL) and driedwith sodium sulfate followed by solvent removal. The crude product wasdissolved in DCM (400 mL) and purified by silica gel chromatography (1kg silica, eluted with 20% ethyl acetate in hexanes (2 L)). Solvent wasremoved by rotary evaporation to provide the title intermediate (81 g)as a light yellow oil.

(c) (4-(benzyloxy)-2-ethyl-5-fluorophenyl)trifluoroborate, potassium

The product of the previous step (81 g, 227 mmol) was mixed with acetone(400 mL) until complete dissolution and methanol (400 mL) was addedfollowed by 3 M potassium hydrogen difluoride in water (379 mL, 1137mmol) and the reaction mixture was stirred at RT. Most of the solventwas removed by rotary evaporation. Water (500 mL) was added and theresulting thick slurry was stirred for 30 min and filtered. The flaskand cake were washed with water (2×100 mL) and the solid was driedovernight. Toluene (400 mL) was added, of which 200 mL was removed byrotary evaporation at 50° C. The reaction mixture was cooled to RT,stirred for 30 min and filtered. The solid was dried to provide thetitle compound (69.7 g, 205 mmol, 90% yield) as a white solid. HPLCMethod A Retention time 10.90 min.

Preparation 9:5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine

(a) 1-Benzyl-4-imino-1,4-dihydropyridin-3-amine

A mixture of pyridine-3,4-diamine (700 g, 6.414 mol) and ACN (15.5 L)was stirred for 80 min from 25° C. to 15° C. A solution of benzylbromide (763 mL, 6.414 mol) in ACN (1 L) was added in 10 min and thereaction mixture was stirred for 1 h at 25° C. and at 20° C. overnight.The reaction mixture was filtered. The reactor and cake were washed withACN (8 L) and warmed to 25° C., and again washed with ACN (8 L) andwarmed to 25° C. The solid was dried on the filter for 3 h undernitrogen, at 50° C. under vacuum for 2 h and then at RT overnight toprovide the HBR salt of the title intermediate (1659 g, 5.922 mol, 92%yield). HPLC Method B Retention time 3.74 min.

(b) 5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine

A solution of 6-bromo-1H-indazole-3-carbaldehyde (558 g, 2.480 mol), theproduct of the previous step (746 g, 2.529 mol) and DMF (4.75 L) wasstirred for 80 min and sodium bisulfite (261 g, 2.504 mol) was addedwith mixing. The reaction mixture was heated to 135° C. and held for 2 hand allowed to cool to RT in about 3 h, cooled to 2° C. and held for 1 hat 0-5° C. The slurry was filtered on a pressure filter by slowfiltration. To the reactor was added DMF (1 L) and the reaction mixturewas cooled to 5° C. The cake was washed and the procedure repeated withanother portion of DMF (4 L). The cake was washed with ACN (1 L) anddried under nitrogen and under vacuum overnight to provide the titlecompound (1080 g, 2.591 mol, 105% yield, 97% purity) as a light yellowsolid. HPLC Method B Retention time 7.83 min.

A mixture of the title compound (1000 g, 2.474 mol) and MeTHF (6 L) washeated to 55° C. and 1 M sodium hydroxide (3.216 L) was added in 5 min.The temperature dropped to 45° C. and the mixture was diluted with coldsodium hydroxide solution. The layers were allowed to separate and theaqueous later was drained. The mixture was cooled to RT and then to 5°C. and held overnight. The mixture was filtered and the reactor and cakewashed with MeTHF (1 L). The resulting beige to yellow solid was driedon the filter for 3 days to provide the title compound (700 g, 1.680mol, 67.9% yield, 97% purity) as a pale yellow solid. HPLC Method BRetention time 7.84 min.

Preparation 10:5-benzyl-2-(6-bromo-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

To a 15 L flask was added5-benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine (350 g,866 mmol) followed by MeTHF (4 L), methanol (2 L) and water (1 L). Theslurry was stirred at 25° C. for 45 min and NaBH₄ (197 g, 5195 mmol) wasadded in two portions. The reaction mixture was stirred at 25° C. for 18h. Water (1 L) was added followed by 20 wt %. sodium chloride solution(2 L) and the reaction mixture was stirred for 30 min and the layersallowed to separate. The aqueous layer was drained; NaOH (1.732 L) wasadded and the reaction mixture was stirred for 30 minutes; the layerswere allowed to separate and the aqueous layer was drained.

The organic layer was combined with the product of a second batch at thesame scale and concentrated to about half the volume by rotaryevaporation at 55° C. The layers were allowed to settle and the aqueouslayer was drained. To the organic layer was added 3M HCl in CPME (1.732L) at 35° C. followed by MeTHF (4 L) and MeOH (4 L) and the mixtureheated to 60° C. to form a thick slurry, cooled to 25° C. in 5 h andheld at that temperature overnight. The slurry was transferred to apressure filter and the wet cake transferred to two tray-driers at 55°C. and dried under vacuum and under nitrogen for 6 h and then at 35° C.for 2 days to afford the 3 HCl salt of the title compound (609 g, 1153mmol, 66.6% yield, 98% purity) as a bristle yellow/beige solid. HPLCMethod B Retention time 5.93 min.

Preparation 11:5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

To a 5 L flask was added cesium carbonate (123 g, 377 mmol) and water(455 mL) with stirring at 22° C., followed by5-benzyl-2-(6-bromo-TH-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine,3HCl (65 g, 126 mmol) and MeOH (1365 mL). The slurry was heated toreflux for 0.5 h and cesium carbonate (127 g, 389 mmol) was addedfollowed by (4-(benzyloxy)-2-ethyl-5-fluorophenyl)trifluoroborate,potassium (52.8 g, 157 mmol). The slurry was purged with nitrogen threetimes, bis(di-tert-butyl(4-dimethylaminophenyl) phosphine)dichloropalladium(II) (8.89 g, 12.56 mmol) was added and the reactionmixture was heated at reflux for 42 h. Additional(4-(benzyloxy)-2-ethyl-5-fluorophenyl)trifluoroborate, potassium (5.28g, 15.7 mmol) and cesium carbonate (16.4 g, 50.3 mmol) were added andthe reaction mixture was stirred at reflux for an additional 18 h andcooled to 25° C.

To the reaction mixture was added 1M HCl in water (502 mL, 502 mmol)followed by water (3 L). The resulting slurry was stirred at 22° C. for1 h, and filtered. The filter cake was rinsed with water (1 L) and driedunder vacuum and under nitrogen to provide the 3HCl salt of the titlecompound (88 g, 132 mmol, 105% yield) which was used directly in thefollowing step. HPLC Method B Retention time 10.07 min.

Preparation 12:5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

A solution of5-benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(70.6 g, 127 mmol) in EtOH (845 mL) and 1.25 M HCl in MeOH (203 mL, 253mmol) was stirred under nitrogen for 10 min with heating to 50° C. andthen 10 wt % Pd/C (8.45 g) was added immediately followed by hydrogengas. The reaction mixture was sealed under hydrogen (50 Psi) at 50° C.for 3 h, filtered through Celite®, and concentrated to 169 mL. Ethylacetate (845 mL) was added, the reaction mixture was concentrated to 169mL, EtOAc (1521 mL) was added and the reaction mixture was stirred at22° C. for 1 h, cooled to 0° C., then held for 1 h and filtered. Thecake was rinsed with EtOAc (100 mL) and dried under vacuum and undernitrogen to afford the 3 HCl salt of the title product (52 g, 107 mmol,70.5% yield). HPLC Method B Retention time 6.06 min.

Preparation 13:5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine

(a) 5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine

A solution of 6-bromo-1H-indazole-3-carbaldehyde (550 g, 2.444 mol),1-benzyl-4-imino-1,4-dihydropyridin-3-amine HBr (721 g, 2.333 mol) andDMAc (2.65 L) was stirred for 60 min and sodium bisulfite (257 g, 2.468mol) was added. The reaction mixture was heated to 135° C. and held for3 h, and allowed to cool to 20° C. and held at 20° C. overnight.Acetonitrile (8 L) was added and the reaction mixture was stirred for 4h at 15° C. The slurry was filtered on a pressure filter at mediumfiltration rate. To the reactor was added ACN (1 L) The cake was washedwith the ACN reactor wash and dried under nitrogen overnight and thenunder vacuum at 50° C. for 24 h to provide the HBr salt of the titlecompound (1264 g, 2.444 mol, 100% yield, 94% purity) as a dense wetbeige/brown solid. HPLC Method B Retention time 8.77 min.

A mixture of the product of the previous step (1264 g, 2.444 mol), MeTHF(6 L) and water (2.75 L) was heated to 65° C. and sodium hydroxide 50 wt% (254 g, 3.177 mol) was added over 5 min and the reaction mixture wasstirred at 65° C. for 1 h, cooled to RT, then to 5° C. and held for 2 h.The slurry was filtered and the reactor and cake were washed with MeTHF(1 L). The resulting beige to yellow solid was dried on the filter undernitrogen for 3 d to provide the title compound (475 g, 1.175 mmol, 48%yield) as a beige/yellow solid. The mother liquor (about 8 L) wasconcentrated to about 2 L, whereupon solids began to crash out. Theslurry was heated to 50° C., held for 2 h, cooled to 5° C. over 2 h,stirred overnight, and filtered. The cake was washed with MeTHF (100 mL)and dried overnight under vacuum at 40° C. to provide additional titlecompound (140 g, 0.346 mol, 14% yield).

A mixture of the total product of the previous step, combined with theproduct of a second batch at the same scale (1500 g, 3.710 mol) andMeTHF (4 L) was stirred at 20° C. for 2 h and filtered. The reactor andcake were washed with MeTHF (1.5 L). The resulting beige to yellow solidwas dried under nitrogen for 3 d to provide the title compound as abeige yellow solid (1325 g, 3.184 mol, 86% yield (overall 68% yield),97% purity). HPLC Method B Retention time 8.77 min

Preparation 14:5-benzyl-2-(6-bromo-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

To a 15 L flask was added5-benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine (440 g,1.088 mol) followed by MeTHF (4.5 L), methanol (2.25 L) and water (1.125L). The slurry was cooled to 20° C., stirred for 1 h, and NaBH₄ (247 g,6.530 mol) was added. The reaction mixture was stirred at 25° C. for 18h. Water (1.125 L) was added followed by 20 wt %. sodium chloridesolution (1.125 L) and the mixture was stirred for 30 min and the layersallowed to separate. The aqueous layer was drained. A premixed solutionof NaOH (522 g) and water (5 L) was added and the reaction mixture wasstirred for 60 min; the layers were allowed to separate and the aqueouslayer was drained. Two additional batches at the same scale wereprepared.

The organic layer from one batch was concentrated under reduced pressurein a 15 L jacketed reactor with the jacket set at 50° C., internaltemperature 20° C. The additional batches were added to the reactor andconcentrated one at a time resulting in a slurry about 6 L in volume.The slurry was heated to 50° C., IPAc (6 L) was added and the mixturewas held at 60° C. for 1.5 h, cooled to 20° C. for 10 h, heated to 60°C. for 50 h, cooled to 20° C. in 5 h, then cooled to 5° C. and held for3 h. The mixture was filtered and the reactor and cake was washed with apremixed solution of IPAc (1 L) and MeTHF (1 L), precooled to 5° C. Thesolids were dried under nitrogen on the filter at 40° C. for 3 d toprovide the title compound (1059 g, 2.589 mol, 79% yield) as anoff-white solid. The material was further dried in a vacuum oven at50-60° C. for 8 h and at 27° C. for 2 d to provide the title compound(1043 g, 2.526 mol, 77% yield, 99% purity). HPLC Method B Retention time6.73 min.

Preparation 15: (4-(Benzyloxy)-2-ethyl-5-fluorophenyl)trifluoroborate,potassium

(a)2-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

A mixture of 1-(benzyloxy)-4-bromo-5-ethyl-2-fluorobenzene (520 g, 1682mmol) and dioxane (5193 mL) was purged with nitrogen and thenbis(pinacolato)diboron (641 g, 2523 mmol) was added followed bypotassium acetate (495 g, 5046 mmol). The reaction mixture was purgedwith nitrogen; Pd(dppf)Cl₂ (41.2 g, 50.5 mmol) was added; the reactionmixture was purged with nitrogen, heated at 103° C. under nitrogen for 5h; and cooled to RT. The reaction mixture was concentrated by vacuumdistillation and partitioned between ethyl acetate (5204 mL) and water(5212 mL). The reaction mixture was filtered through Celite; the organiclayer was washed with brine (2606 mL) followed by solvent removal byvacuum distillation to provide crude product as a thick black oil (˜800g).

The crude product was dissolved in DCM (1289 mL) and purified by silicagel chromatography (2627 g silica preslurried in hexane, eluted with 20%ethyl acetate in hexanes (10.35 L)). Solvent was removed by vacuumdistillation to yield a light yellow oil (600 g). HPLC Method CRetention time 33.74 min.

(b) (4-(benzyloxy)-2-ethyl-5-fluorophenyl)trifluoroborate, potassium

The product of the previous step (200 g, 561 mmol) was mixed withacetone (1011 mL) until complete dissolution and methanol (999 mL) wasadded followed by 3 M potassium hydrogen difluoride (307 g, 3930 mmol)dissolved in water (1310 mL). The reaction mixture was stirred for 3.5h. Most of the organic solvent was removed by vacuum distillation. Water(759 mL) was added and the resulting thick slurry was stirred for 30 minand filtered. The cake was washed with water (506 mL) and the solidswere dried on the filter for 30 min. The solids were slurried in acetone(1237 mL) and stirred for 1 h. The resulting slurry was filtered and thesolids washed with acetone (247 mL). The acetone solution wasconcentrated by vacuum distillation, and a constant volume (2 L) wasmaintained by slow addition of toluene (2983 mL) until all acetone andwater had been distilled. The toluene solution was distilled to a thickyellow slurry by rotary evaporation, during which time the productsprecipitated as white solids. An additional portion of toluene (477 mL)was added to the mixture and stirred for 1 h. The mixture was thenfiltered and rinsed with toluene (179 mL) and dried under vacuum at 50°C. for 24 h to provide the title compound (104 g, 310 mmol, 55% yield)as a free-flowing, fluffy, slightly off-white solid. HPLC Method CRetention time 27.71 min.

Preparation 16:5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

(a)5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

A mixture of bis(pinacolato)diboron (250 g, 984 mmol) and IPA (1.88 L)was stirred to dissolution and then a solution of potassium hydrogendifluoride (538 g, 6.891 mol) in water (2.31 L) was added portion-wiseover 10 min. The reaction mixture was stirred for 1 h and filtered. Thegel-like solids were slurried with water (1.33 L) until the mixtureformed a clear hydrogel and then for another 45 min. The resultingsolids/gel were filtered, then reslurried in acetone (1.08 L), filtered,air dried on the filter for 30 min and dried overnight to provide afluffy white solid (196.7 g).

To a 5 L flask was added5-benzyl-2-(6-bromo-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(135 g, 331 mmol),(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-trifluoroborate, potassium (133g, 397 mmol), and the white solid product of the previous step (40.5 g)followed by MeTHF (1.23 L) and MeOH (1.75 L). The resulting slurry wasdegassed three times with nitrogen. To the slurry was added a degassedsolution of cesium carbonate (431 g, 1.323 mol) in water (1.35 L). Theslurry was degassed twice, Pd (amphos)₂Cl₂ (11.71 g, 16.53 mmol) wasadded, the slurry was again degassed twice and the reaction mixture wasstirred at 67° C. overnight and cooled to 20° C. The layers wereseparated and back extracted with MeTHF (550 mL). The organic layerswere combined and concentrated by rotary evaporation until solidsprecipitated. MeTHF (700 mL) was added and the reaction mixture wasstirred at 65° C. The layers were separated and the aqueous phase backextracted with MeTHF (135 mL). The organic phases were combined andconcentrated to about 300 mL resulting in a thick orange slurry. To theslurry was added MeOH (270 mL) followed by 1M HCl (1.325 L) at 20° C.with rapid stirring. The reaction mixture was stirred for 5 min andwater (1 L) was added and the resulting slurry was stirred for 1 h. Thesolids were filtered, washed with water (150 mL), dried on the filterfor 10 min and at 45° C. under nitrogen for 16 h to provide the 2 HClsalt of the title compound (221.1 g, 351 mmol, 92.2% purity) as a lightyellow solid. HPLC Method C retention time 23.41 min.

Preparation 17:5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To a 1 L flask was added5-benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine,2 HCl (40 g, 63.4 mmol) as a slurry in ethanol (348 mL) and 1.25 M HClin MeOH (101 mL) and water (17.14 mL). The reaction mixture was degassedwith nitrogen for 5 min and 10 wt % Pd/C, 50 wt % H₂O (4.05 g, 1.903mmol) was added. The reactor was sealed, purged with H₂, pressurized to1-2 psi. warmed to 50° C., and the reaction mixture was stirredovernight and filtered through Celite. The reactor and filter werewashed with methanol (100 mL).

The filtered solution was combined with the product of a second batch atthe 98 mmol scale and concentrated to 390 g. EtOAc (2.04 L) was addedslowly with stirring and then the solution was cooled to 5° C. withstirring. Solids were filtered, washed with EtOAc (510 mL), and driedovernight at 45° C. under nitrogen to provide the 2 HCl salt of thetitle compound (58 g, 80% yield) as an off-white solid. HPLC Method Cretention time 12.83 min.

Example 1:5-Ethyl-2-fluoro-4-(3-(5-(1-methylazetidin-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

(a)4-(3-(5-(azetidin-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol

A mixture of5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolHCl (300 mg, 0.795 mmol), tert-butyl 3-oxoazetidine-1-carboxylate (272mg, 1.590 mmol) and acetic acid (0.137 ml, 2.385 mmol) in a mixture ofTHF (6 mL) and DMF (2 mL) was heated at 40° C. for 30 min. The reactionmixture was cooled to RT, treated with sodium triacetoxyborohydride (505mg, 2.385 mmol), and heated at 40° C. for 2 h. The reaction mixture wascombined with a parallel reaction at the 0.132 mmol scale andconcentrated. The resulting residue was partitioned between EtOAc (200mL) and saturated ammonium chloride (30 mL). The organic layer waswashed with water (2×20 mL), dried over sodium sulfate, filtered,concentrated, and purified by silica gel chromatography (24 g silicagel, 0-15% MeOH/DCM). Desired fractions were combined and concentratedto give a white soft solid.

The solid was treated with 4 N HCl in 1,4-dioxane (3.97 mL) and water (1mL) at RT for 2 h, concentrated, and freeze dried to give the HCl saltof the title intermediate (388 mg, 0.768 mmol, 83% yield) as a whitesolid. (m/z): [M+H]⁺ calcd for C₂₄H₂₅FN₆O 433.21 found 433.

(b)5-Ethyl-2-fluoro-4-(3-(5-(1-methylazetidin-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To a solution of the product of the previous step (259.4 mg, 0.513 mmol)in MeOH (7 ml) at RT was added formaldehyde solution, 37% in water(0.076 mL, 1.026 mmol). The reaction mixture was stirred for 5 min andthen sodium cyanoborohydride (129 mg, 2.053 mmol) was added and themixture left overnight. The next day sodium borohydride (194 mg, 5.13mmol) was added at RT. After 1 h, the reaction was quenched by the slowaddition of acetic acid (5 mL) and water (2 mL). The reaction mixturewas stirred for 30 min, concentrated, and additional water (3 mL) wasadded. The reaction mixture was filtered, purified by preparative HPLC,and freeze dried to provide the TFA salt of the title compound (132 mg)as a yellowish solid. (m/z): [M+H]⁺ calcd for C₂₅H₂₇FN₆O 447.22 found447. ¹H NMR (400 MHz, Methanol-d4) δ 8.17 (dd, J=8.5, 0.9 Hz, 1H), 7.56(dd, J=1.4, 0.8 Hz, 1H), 7.33 (dd, J=8.5, 1.4 Hz, 1H), 6.94 (d, J=11.6Hz, 1H), 6.90 (d, J=8.9 Hz, 1H), 4.58-4.43 (m, 1H), 4.41-4.28 (m, 1H),4.23-3.97 (m, 2H), 3.81-3.67 (m, 3H), 3.00 (s, 3H), 2.97-2.88 (m, 4H),2.53 (q, J=7.5 Hz, 2H), 1.05 (t, J=7.5 Hz, 3H).

Example 2:4-(3-(5-(azetidin-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol

To a solution of5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(50 mg, 0.132 mmol) and tert-butyl 3-oxoazetidine-1-carboxylate (68.0mg, 0.397 mmol) in methanol (2 mL) was added sodium cyanoborohydride(50.0 mg, 0.795 mmol) and the reaction mixture was stirred at RT,dissolved in 5 mL of 2:1 acetic acid:water (5 mL) and purified bypreparative HPLC. The product fractions were combined and the solventwas evaporated. To the pure dry product was added ACN (1 mL) and 4 N HClin dioxane (1 mL). The reaction mixture was stirred at RT for 30 minconcentrated and purified by preparative HPLC to provide the TFA salt ofthe title compound (20 mg). (m/z): [M+H]⁺ calcd for C₂₄H₂₅FN₆O 433.21found 433.

Example 3:5-ethyl-2-fluoro-4-(3-(5-(1-isopropylazetidin-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To a solution of4-(3-(5-(azetidin-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol(15 mg, 0.035 mmol) and acetone (10.07 mg, 0.173 mmol) in MeOH (2.0 ml)was added sodium cyanoborohydride (17.44 mg, 0.277 mmol) and thereaction mixture was stirred at RT overnight, concentrated under vacuumand purified by preparative HPLC to provide the TFA salt of the titlecompound (10.4 mg). (m/z): [M+H]⁺ calcd for C₂₇H₃₁FN₆O 475.25 found475.1.

Example 4:4-(3-(5-(1-(sec-butyl)azetidin-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol

Using a process similar to that of Example 3 at the 0.045 mmol scalewith reagent 2-butanone in place of acetone, the TFA salt of the titlecompound (10 mg) was prepared. (m/z): [M+H]⁺ calcd for C₂₈H₃₃FN₆O 489.27found 489.2.

Example 5:4-(3-(5-(1-cyclopropylazetidin-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol

To a solution of4-(3-(5-(azetidin-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol(349 mg, 0.807 mmol), [(1-ethoxycyclopropyl)oxy]-trimethylsilane (0.811mL, 4.03 mmol), and acetic acid (0.185 ml, 3.23 mmol) in methanol (4.03mL) was added sodium cyanoborohydride (507 mg, 8.07 mmol) in methanol(4.03 mL) was added. The reaction mixture was stirred at 65° C. for 2 h,concentrated by rotary evaporation, and purified by preparative HPLC.Fractions were combined to provide the TFA salt of the title compound(62 mg). (m/z): [M+H]⁺ calcd for C₂₇H₂₉FN₆O 473.24 found 473.2.

Example 6:5-Ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To a solution of5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolHCl (0.80 g, 1.93 mmol), acetic acid (0.33 mL, 5.80 mmol), and1-methylpiperidin-4-one (0.29 mL, 2.32 mmol) in DMF (30 mL) was addedsodium triacetoxyborohydride (1.229 g, 5.80 mmol). The reaction mixturewas stirred at RT for 48 h, concentrated, and purified by preparativeHPLC to provide the title compound (612 mg). (m/z): [M+H]⁺ calcd forC₂₇H₃₁FN₆O 475.25 found 475.1. ¹H NMR (400 MHz, Methanol-d₄) δ 8.19 (dd,J=8.5, 1.0 Hz, 1H), 7.51 (d, J=1.4 Hz, 1H), 7.26 (dd, J=8.5, 1.5 Hz,1H), 6.94 (d, J=11.7 Hz, 1H), 6.89 (d, J=8.9 Hz, 1H), 4.22 (s, 2H),3.75-3.61 (m, 2H), 3.53-3.37 (m, 4H), 3.22-3.08 (m, 1H), 3.07-3.00 (m,2H), 2.91 (s, 3H), 2.52 (q, J=7.5 Hz, 2H), 2.43-2.30 (m, 3H), 2.19-2.01(m, 3H), 1.05 (t, J=7.5 Hz, 3H).

Example 7:4-(3-(5-(2-(dimethylamino)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol

(a) tert-Butyl(2-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)carbamate

To a suspension of5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolHCl (600 mg, 1.45 mmol) in DMF (20 mL) were added tert-butyl(2-oxoethyl)carbamate (277 mg, 1.74 mmol) and acetic acid (0.25 mg, 4.35mmol) followed by sodium triacetoxyborohydride (922 mg, 4.35 mmol) inportions over several min and the reaction mixture was stirred at RT for96 h. The reaction mixture was concentrated by rotary evaporation andpurified by preparative HPLC to provide the TFA salt of the titleintermediate (364 mg).

(b)4-(3-(5-(2-aminoethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol

To the product of the previous step (364 mg, 0.57 mmol) were added 4 MHCl in dioxane (3 mL) and water (0.1 mL). The reaction mixture wasstirred at RT for 30 min, concentrated by rotary evaporation, evaporatedwith EtOAc (3×5 mL) by rotary evaporation, and dried under high vacuumto provide the HCl salt of the title intermediate (283 mg) which wasused directly in the next step.

(c)4-(3-(5-(2-(dimethylamino)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol

To a solution of the product of the previous step (283 mg) in MeOH (11mL) at RT was added formaldehyde solution, 37% in water (0.171 mL, 2.30mmol). The reaction mixture was stirred for 5 min and then sodiumcyanoborohydride (252 mg, 4.02 mmol) was added. One hour 15 min later,sodium borohydride (152 mg, 4.02 mmol) was added.

After 1 h, the reaction mixture was concentrated by rotary evaporationand purified by preparative HPLC to provide the TFA salt of the titlecompound (141 mg) as a yellow powder. (m/z): [M+H]⁺ calcd for C₂₅H₂₉FN₆O449.24 found 449. ¹H NMR (400 MHz, Methanol-d4) δ 8.16 (dd, J=8.5, 0.9Hz, 1H), 7.55 (d, J=1.3 Hz, 1H), 7.32 (dd, J=8.4, 1.4 Hz, 1H), 6.94 (d,J=11.6 Hz, 1H), 6.90 (d, J=9.0 Hz, 1H), 3.89 (s, 2H), 3.40 (dd, J=6.5,5.0 Hz, 2H), 3.12 (t, J=5.7 Hz, 2H), 3.06 (t, J=5.8 Hz, 2H), 2.98-2.86(m, 8H), 2.52 (q, J=7.5 Hz, 2H), 1.05 (t, J=7.5 Hz, 3H).

Example 8:5-Ethyl-2-fluoro-4-(3-(5-(2-((3-methoxycyclobutyl)amino)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

(a) tert-Butyl(2-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)carbamate

To a suspension of5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolHCl (600 mg, 1.45 mmol) in DMF (20 mL) were added tert-butyl(2-oxoethyl)carbamate (277 mg, 1.74 mmol) and acetic acid (0.25 mg, 4.35mmol) followed by sodium triacetoxyborohydride (922 mg, 4.35 mmol) inportions over several min and the reaction mixture was stirred at RT for96 h. The reaction mixture was concentrated and purified by preparativeHPLC (10-70% ACN/Water). to provide the TFA salt of the titleintermediate (507 mg).

(b)4-(3-(5-(2-aminoethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol

The product of the previous step (505 mg, 0.80 mmol) was dissolved indioxane (8 mL) and water (1.6 mL) and then 4 M HCl in dioxane (8 mL, 32mmol) was added. The reaction mixture was stirred at RT for 20 min,frozen, and lyophilized to provide the HCl salt of the titleintermediate which was used directly in the next step.

(c)5-Ethyl-2-fluoro-4-(3-(5-(2-((3-methoxycyclobutyl)amino)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

The product of the previous step (393 mg, 0.80 mmol) and acetic acid(0.14 mL, 2.39 mmol) were dissolved in DMF (10 mL), then3-methoxycyclobutane-1-one (0.094 mL, 0.88 mmol) was added. The reactionmixture was stirred at RT for 30 min and sodium triacetoxyborohydride(507 mg, 2.39 mmol) was added. The reaction mixture was stirred at RTovernight, concentrated and purified by preparative HPLC to provide theTFA salt of the title intermediate (56 mg). (m/z): [M+H]⁺ calcd forC₂₈H₃₃FN₆O₂ 505.26 found 505.3.

Example 9:5-ethyl-4-(3-(5-(2-(ethyl(methyl)amino)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-2-fluorophenol

(a) tert-butyl(2-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethyl)(methyl)carbamate

Acetic acid (0.166 mL, 2.90 mmol), tert-butylmethyl(2-oxoethyl)carbamate (201 mg, 1.160 mmol), and5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,HCl (400 mg, 0.966 mmol) were combined in DMF (3.65 mL). Sodiumtriacetoxyborohydride (615 mg, 2.90 mmol) was added portion wise overfive minutes. The reaction mixture was stirred overnight and dilutedwith EtOAc (50 mL). The organic solution was washed with sat. NaHCO₃(2×20 mL). The organic phase was collected, dried (MgSO4), andconcentrated under vacuum. The crude residue was purified by silica gelchromatography (0% to 15% MeOH in DCM). Pure fractions were combined andconcentrated to provide the title intermediate (491 mg) as a colorless,amorphous solid. (m/z): [M+H]⁺ calcd for C₂₉H₃₅FN₆O₃ 535.28 found 536.

(b)5-ethyl-2-fluoro-4-(3-(5-(2-(methylamino)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

The product of the previous step (0.491 g, 0.918 mmol) was dissolved indioxane (4.59 mL) and water (4.59 mL) and 4 N HCl in dioxane (4.59 mL,18.36 mmol) was added slowly over 5 min. The reaction mixture wasstirred for 1 h, diluted with water (20 mL), freeze dried at −78° C.,and lyophilized to provide the di-HCL salt of the title intermediate(413 mg).

(c)5-ethyl-4-(3-(5-(2-(ethyl(methyl)amino)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-2-fluorophenol

To the product of the previous step (0.2 g, 0.394 mmol) dissolved inMeOH (1.971 mL) was added acetaldehyde (0.11 mL, 1.971 mmol) followed bysodium cyanoborohydride (248 mg, 3.94 mmol). The reaction mixture wasstirred overnight, dissolved in 2:1 acetic acid:water, syringe filtered,and purified by preparative HPLC. Pure fractions were combined andlyophilized to provide the TFA salt of the title compound (25 mg).(m/z): [M+H]⁺ calcd for C₂₆H₃₁FN₆O₃ 436.28 found 436.2.

Example 10:4-(3-(5-(2-(sec-butyl(methyl)amino)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol

Following the general procedure of Example 9 using butan-2-one (0.177mL, 1.971 mmol) in place of acetaldehyde in step (c) the TFA salt of thetitle compound was prepared (66 mg). (m/z): [M+H]⁺ calcd for C₂₈H₃₅FN₆O₃491.29 found 492.

Example 11:(S)-5-ethyl-2-fluoro-4-(3-(5-((1-methylpyrrolidin-2-yl)methyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

(a) tert-butyl(S)-2-((2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)methyl)pyrrolidine-1-carboxylate

To a suspension of5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(50 mg, 0.132 mmol) and (S)-tert-butyl 2-formylpyrrolidine-1-carboxylate(34.3 mg, 0.172 mmol) in methanol (1.34 mL) was added sodiumcyanoborohydride (33.3 mg, 0.530 mmol) and the reaction mixture wasstirred at 25° C. overnight. Additional sodium cyanoborohydride (33.3mg, 0.530 mmol) was added and the reaction mixture was heated at 70° C.for 30 min. The following day, two additional portions of (S)-tert-butyl2-formylpyrrolidine-1-carboxylate (34.3 mg, 0.172 mmol) were added, eachfollowed by heating added at 70° C. for 1 h. The reaction mixture wasdissolved in 8:2 DCM:methanol (8 mL) and purified by silica gelchromatography (100% DCM 15 min, 0-5% DCM:methanol, 20 min, 5%DCM:methanol, 20 min). Fractions were combined and concentrated toprovide the title intermediate as a white waxy solid (167 mg). (m/z):[M+H]⁺ calcd for C₃₁H₃₇FN₆O₃ 561.29 found 561.3.

(b)(S)-5-ethyl-2-fluoro-4-(3-(5-(pyrrolidin-2-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To the product of the previous step (167 mg, 0.298 mmol) was added DCM(14.9 mL) followed by TFA (14.9 mL) and the reaction mixture was stirredfor 1 h, concentrated, and dissolved in 4:1 water:acetic acid (8 mL)with 8 drops of methanol and purified by preparative HPLC. Fractionswere combined and concentrated to provide the TFA salt of the titleintermediate (70 mg) as a glassy white solid. (m/z): [M+H]⁺ calcd forC₂₆H₂₉FN₆O 461.24 found 461.1.

(c)S)-5-ethyl-2-fluoro-4-(3-(5-((1-methylpyrrolidin-2-yl)methyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To a solution of the product of the previous step (70 mg, 0.152 mmol)and formaldehyde solution 37% in water (0.023 mL, 0.304 mmol) inmethanol (15.2 mL) was added sodium cyanoborohydride (38.2 mg, 0.608mmol) and the reaction mixture was stirred at 25° C. overnight. Anadditional portion of formaldehyde (0.023 mL, 0.304 mmol) was added andthe reaction mixture was stirred at 25° C. overnight and concentrated.Methanol (1.52 mL) and sodium cyanoborohydride (382 mg, 6.08 mmol) wereadded; the reaction mixture was stirred for 3 h; additional sodiumcyanoborohydride (382 mg, 6.08 mmol) was added; the reaction mixture wasstirred at 25° C. over the weekend; concentrated; dissolved 1:1 aceticacid:water (4 mL), filtered and purified by preparative HPLC to providethe TFA salt of the title compound (36.4 mg). (m/z): [M+H]⁺ calcd forC₂₇H₃₁FN₆O 475.25 found 475.2.

Example 12:4-(3-(5-(3-(dimethylamino)-2-fluoropropyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol

(a) tert-butyl(3-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-2-fluoropropyl)carbamate

To a solution of DIPEA (0.505 mL, 2.90 mmol) and5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,HCl (400 mg, 0.966 mmol) in DMF (2.416 mL) was added dropwise a solutionof tert-butyl (3-bromo-2-fluoropropyl)carbamate (248 mg, 0.966 mmol) inDMF (2.416 mL). The reaction mixture was stirred at RT overnight.Additional tert-butyl (3-bromo-2-fluoropropyl)carbamate (248 mg, 0.966mmol) was added and the reaction mixture was stirred overnight,concentrated under vacuum, and purified by silica gel chromatography(MeOH:DCM) to provide the title intermediate (286 mg, 0.518 mmol, 54%yield). (m/z): [M+H]⁺ calcd for C₂₉H₃₄F₂N₆O₃ 553.27 found 553.

(b)4-(3-(5-(3-amino-2-fluoropropyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol

The product of the previous step (0.286 g, 0.518 mmol) was dissolved indioxane (2.15 mL) and water (0.48 mL) and 4 M HCl in dioxane (2.15 mL,8.60 mmol) was added slowly over 5 min and the reaction mixture wasstirred at RT for 30 min, frozen, and lyophilized to provide the HClsalt of the title intermediate (261 mg). (m/z): [M+H]⁺ calcd forC₂₄H₂₆F₂N₆O 453.21 found 453.

(c)4-(3-(5-(3-(dimethylamino)-2-fluoropropyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol

The product of the previous step (0.261 g, 0.497 mmol) and formaldehydesolution 37% in water (0.083 mL, 1.043 mmol) were combined in MeOH (4.97mL). Sodium cyanoborohydride (0.156 g, 2.484 mmol) was added and thereaction mixture was stirred at RT for several hours. Sodium borohydridewas added. The reaction mixture was concentrated and purified bypreparative HPLC. Pure fractions were combined and lyophilized toprovide the TFA salt of the title compound (30 mg). (m/z): [M+H]⁺ calcdfor C₂₆H₃₀F₂N₆O 481.24 found 481.

Example 13:(S)-5-Ethyl-2-fluoro-4-(3-(5-(morpholin-3-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To a solution of5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolTFA (100 mg, 0.203 mmol) and tert-butyl(R)-3-formylmorpholine-4-carboxylate (285 mg, 1.325 mmol) in MeOH (5mL), was added sodium cyanoborohydride (167 mg, 2.65 mmol) and thereaction mixture was stirred at RT overnight.

The reaction mixture was concentrated and TFA (3 mL) was added at 0° C.After 30 min, the reaction mixture was concentrated and purified bypreparative HPLC (2-70% ACN/Water) to provide the TFA salt of the titlecompound (55.2 mg). (m/z): [M+H]+ calcd for C₂₆H₂₉FN₆O₂ 477.23 found477.1. ¹H NMR (400 MHz, Methanol-d4) δ 8.16 (dd, J=8.5, 0.9 Hz, 1H),7.55 (d, J=1.2 Hz, 1H), 7.31 (dd, J=8.5, 1.4 Hz, 1H), 6.94 (d, J=11.6Hz, 1H), 6.90 (d, J=9.0 Hz, 1H), 4.04 (ddd, J=15.8, 12.7, 3.4 Hz, 2H),3.97-3.72 (m, 4H), 3.66 (td, J=8.2, 7.3, 3.1 Hz, 1H), 3.57 (dd, J=12.5,9.1 Hz, 1H), 3.35-3.32 (m, 1H), 3.28-3.12 (m, 2H), 3.07-2.96 (m, 1H),2.96-2.90 (m, 2H), 2.88 (d, J=7.3 Hz, 2H), 2.52 (q, J=7.5 Hz, 2H), 1.05(t, J=7.5 Hz, 3H).

Example 14:(R)-5-Ethyl-2-fluoro-4-(3-(5-(morpholin-3-ylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

Using a process similar to that of Example 13 at the 0.159 mmol scale,the TFA salt of the title compound (29.1 mg) was prepared. (m/z): [M+H]⁺calcd for C₂₆H₂₉FN₆O₂ 477.23 found 477.1.

Example 15:(S)-5-ethyl-2-fluoro-4-(3-(5-(2-(2-methylpyrrolidin-1-yl)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

(a)4-(3-(5-(2,2-dimethoxyethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)-5-ethyl-2-fluorophenol

To a mixture of5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolHCl (200 mg, 0.483 mmol) and 2,2-dimethoxyacetaldehyde (0.146 mL, 0.966mmol) in MeOH (4.83 mL) was added sodium cyanoborohydride (121 mg, 1.933mmol) and the reaction mixture was stirred overnight at RT,concentrated, and purified by silica gel chromatography (10% MeOH inDCM) to provide the title intermediate, (210 mg). (m/z): [M+H]⁺ calcdfor C₂₅H₂₈FN₅O₃ 466.22 found 466.

(b)2-(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)ethane-1,1-diol

The product of the previous step (210 mg, 0.0.451 mmol) was dissolved inMeTHF (4 mL) and 3 N HCl in water (4 mL, 12 mmol) was added. Thereaction mixture was stirred for 4 d, concentrated, dissolved in 1:1acetic acid:water, and purified by preparative HPLC. Pure fractions werecombined and lyophilized to provide the title intermediate (150 mg)(m/z): [M+H]⁺ calcd for C₂₃H₂₄FN₅O₃ 438.19 found 438.

(c)(S)-5-ethyl-2-fluoro-4-(3-(5-(2-(2-methylpyrrolidin-1-yl)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To a mixture of the product of the previous step (20 mg, 0.046 mmol) and(S)-2-methylpyrrolidine (19 mg, 0.229 mmol) in MeOH (0.5 mL) was addedsodium cyanoborohydride (28.7 mg, 0.457 mmol) in MeOH (0.5 mL). Thereaction mixture was stirred overnight, dissolved in 2:1 aceticacid:water and purified by preparative HPLC to provide the TFA salt ofthe title compound (6 mg, 0.0084 mmol, 18% yield). (m/z): [M+H]⁺ calcdfor C₂₈H₃₃FN₆O 489.27 found 489.2.

Example 16:5-Ethyl-2-fluoro-4-(3-(5-(2-(pyrrolidin-1-yl)ethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To a solution of5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(30 mg, 0.079 mmol) in DMF (500 μL) was added1-(2-bromoethyl)pyrrolidine (21.23 mg, 0.119 mmol) and DIPEA (69.2 μL,0.397 mmol). The reaction mixture was capped and stirred at RT for 1 h,concentrated, dissolved in 1:1 acetic acid:water, and purified bypreparative HPLC to provide the TFA salt of the title compound (18 mg,0.026 mm, 32% yield). (m/z): [M+H]+ calcd for C₂₇H₃₁FN₆O 475.25 found475.2.

Using similar synthetic methods, the compounds of Tables 1-19 wereprepared. In the following tables, a blank in any column indicates ahydrogen atom, a * in a structure heading a table indicates a chiralcenter, and the notation (R) or (S) in front of a substituent denotesthe configuration of the carbon atom to which the substituent isattached.

TABLE 1

Calc Found Ex No. R⁸ R³ Formula [M + H]⁺ [M + H]⁺ 1-1 —(CH₂)₂OHC₂₆H₂₉FN₆O₂ 477.23 477.2 1-2 —CH₃ C₂₅H₂₇FN₆O 447.22 447 1-3 —C₂H₅C₂₆H₂₉FN₆O 461.24 461.2 1-4 —(CH₂)₃SCH₃ C₂₈H₃₃FN₆OS 521.24 521.2 1-5

C₂₉H₃₀FN₇O 512.25 512.3 1-6 —(CH₂)₂OCH₃ C₂₇H₃₁FN₆O₂ 491.25 491.1 1-7—(CH₂)₃OCH₃ C₂₈H₃₃FN₆O₂ 505.27 505.2 1-8 —(CH₂)₂CN C₂₇H₂₈FN₇O 486.23486.1 1-9 —(CH₂)₂SCH₃ C₂₇H₃₁FN₆OS 507.23 507.2 1-10 cBu C₂₈H₃₁FN₆O487.25 487.2 1-11 —CH₂iPr C₂₈H₃₃FN₆O 489.27 489.2 1-12 oxetan-3-ylC₂₇H₂₉FN₆O₂ 489.23 489.1 1-13 —(CH₂)₂CH(CH₃)SCH₃ C₂₉H₃₅FN₆OS 535.26535.1 1-14 —CH₃ —CH₃ C₂₆H₂₉FN₆O 461.24 461 1-15

C₂₈H₃₁FN₆O₃S 551.22 551.1 1-16 tetrahydropyran-4-yl C₂₉H₃₃FN₆O₂ 517.27517.3 1-17

C₂₉H₃₃FN₆O₂ 517.27 517.2 1-18 —CH₂cPr C₂₈H₃₁FN₆O 487.25 487.2

TABLE 2

Calc Found Ex. No. R⁸ R⁴, R⁵, R^(x1), R^(x2) Formula [M + H]⁺ [M + H]⁺2-1 —CH₃ R⁵ = —C(O)OCH₃ C₂₉H₃₃FN₆O₃ 533.26 533 2-2 R⁴ = —C(O)OC₂H₅C₂₉H₃₃FN₆O₃ 533.26 533.1 2-3 C₂₆H₂₉FN₆O 461.24 461.2 2-4 R⁵ = (R)CH₃C₂₇H₃₁FN₆O 475.25 475.2 2-5 R⁵ = (R)CH₃ C₂₈H₃₃FN₆O 489.27 489 R^(x1) =(R)CH₃ 2-6 cBu C₃₀H₃₅FN₆O 515.29 515.2 2-7 —CH₂iPr C₃₀H₃₇FN₆O 517.30517.2 2-8

C₃₀H₃₅FN₆O₃S 579.25 579.2 2-9 oxetan-3-yl C₂₉H₃₃FN₆O₂ 517.27 517.2 2-10—CH₂-phenyl C₃₃H₃₅FN₆O 551.29 551.2 2-11 —(CH₂)₂CN C₂₉H₃₂FN₇O 514.27514.2 2-12 tetrahydropyran-4-yl C₃₁H₃₇FN₆O₂ 545.30 545.3 2-13 cPrC₂₉H₃₃FN₆O 501.27 501.2 2-14 oxetan-3-yl R⁴ and R^(x2) form C₃₁H₃₄F₂N₆O₂561.27 561.2 —(CH₂)₂— 2-15 tetrahydropyran-4-yl R⁴ and R^(x2) formC₃₃H₃₈F₂N₆O₂ 589.30 589.2 —(CH₂)₂— 2-16 —CH₂iPr R⁴ and R^(x2) formC₃₂H₃₆F₂N₆O 559.29 559.2 —(CH₂)₂— 2-17

R⁴ and R^(x2) form —(CH₂)₂ C₃₃H₃₈F₂N₆O₂ 589.30 589.2 2-18 —C₂H₅C₂₈H₃₃F₂N₆O 489.27 489.2 2-19 —CH₂CH₂F C₂₈H₃₂F₂N₆O 507.26 507.3 2-20 iPrC₂₉H₃₅FN₆O 503.29 503.3

TABLE 3

Ex Calc Found No. R⁸ R², R³, R⁴, R⁷ * Formula [M + H]⁺ [M + H]⁺ 3-1

C₂₇H₂₈FN₇O 486.23 486.1 3-2 cBu R⁷ = —CH₃ C₂₈H₃₃FN₆O 489.27 489.2 3-3iPr R⁷ = —CH₃ C₂₇H₃₃FN₆O 477.27 477.2 3-4 —CH₃ R³ = —CH₃ C₂₇H₃₃FN₆O477.27 477.1 R⁴ = —CH₃ R⁷ = —CH₃ 3-5 —CH₃ R³ = —CH₃ (S) C₂₆H₃₁FN₆O463.25 463.2 R⁷ = —CH₃ 3-6

C₂₇H₃₁FN₆O₂ 491.25 491.2 3-7 C₂₃H₂₅FN₆O 421.21 421.1 3-8 —CH₃ R² and R³form C₂₈H₃₃FN₆O 489.27 489.2 —(CH₂)₃— R⁷ = —CH₃ 3-9 R³ = —CH₃ (S)C₂₄H₂₇FN₆O 435.22 435 3-10 —(CH₂)₂SCH₃ C₂₆H₃₁FN₆OS 495.23 495.1 3-11 cBuC₂₇H₃₁FN₆O 475.25 475.2 3-12 —(CH₂)₃SCH₃ C₂₇H₃₃FN₆OS 509.24 509.1 3-13—(CH₂)₂CN C₂₆H₂₈FN₇O 474.23 474.1 3-14

C₂₉H₃₅FN₆O 503.29 503.2 3-15 —C₂H₅ C₂₅H₂₉FN₆O 449.24 449.2 3-16 —CH₃C₂₄H₂₇FN₆O 435.22 435.1 3-17 R³ = —CH₃ C₂₅H₂₉FN₆O 449.24 449.1 R⁴ = —CH₃3-18 —(CH₂)₂CH(CH₃)SCH₃ C₂₈H₃₅FN₆OS 523.26 523.2 3-19 R³ = (R)C₂₅H₂₉FN₆O₂ 465.23 465 —CH((R)CH₃)OH 3-20

C₂₇H₃₁FN₆O2 491.25 491.2 3-21 R² = —CH₃ C₂₅H₂₉FN₆O 449.24 449.2 R⁴ =—CH₃ 3-22

R⁷ = —CH₃ C₂₉H₃₅FN₆O₂ 519.28 519 3-23

C₂₈H₃₁F₃N₆O 525.25 525.2 3-24

C₂₈H₃₃FN₆O 489.27 489.2 3-25

C₂₈H₃₀FN₇O 500.25 500.2 3-26

C₂₉H₃₅FN₆O₂ 519.28 519.2 3-27 —CH₂C(CH₃)₂CH₂OC₂H₅ C₃₀H₃₉FN₆O₂ 535.31535.3 3-28 —CH₃ R³ = (R)CH₂OH C₂₆H₃₁FN₆O₂ 479.25 479 R⁷ = —CH₃ 3-29 —CH₃R³ = (S)CH₂OH C₂₆H₃₁FN₆O₂ 479.25 479 R⁷ = —CH₃ 3-30

C₂₈H₃₃FN₆O₂ 505.27 505.2 3-31 tetrahydropyran-4-yl R⁷ = —CH₃ C₂₉H₃₅FN₆O₂519.28 519 3-32 oxetan-3-yl R⁷ = —CH₃ C₂₇H₃₁FN₆O₂ 491.25 491.2 3-33—CH₂iPr R⁷ = —CH₃ C₂₈H₃₃FN₆O 489.27 489 3-34 cPr R⁷ = —CH₃ C₂₇H₃₁FN₆O475.25 475.3 3-35 pyridin-4-yl R⁷ = —CH₃ C₂₉H₃₀FN₇O 512.25 512.2

TABLE 4

Ex. Calc Found No. R⁸ * Formula [M + H]⁺ [M + H]⁺ 4-1 (S) C₂₆H₂₉FN₆O461.24 461.2 4-2 (R) C₂₆H₂₉FN₆O 461.24 461.2 4-3 —CH₃ (R) C₂₇H₃₁FN₆O475.25 475.2 4-4 —C₂H₅ (R) C₂₈H₃₃FN₆O 489.27 489.2 4-5 —CH₂iPr (R)C₃₀H₃₇FN₆O 517.30 517.2 4-6 cBu (R) C₃₀H₃₅FN₆O 515.29 515.2 4-7 iPr (R)C₂₉H₃₅FN₆O 503.29 503.2 4-8 oxetan-3-yl (R) C₂₉H₃₃FN₆O₂ 517.27 517.2 4-9—CH₂iPr (S) C₃₀H₃₇FN₆O 517.30 517.2 4-10 —cBu (S) C₃₀H₃₅FN₆O 515.29515.2 4-11 tetrahydropyran- (S) C₃₁H₃₇FN₆O₂ 545.30 545.2 4-yl 4-12 —C₂H₅(S) C₂₈H₃₃FN₆O 489.27 489.2 4-13 —CH₂cPr (R) C₃₀H₃₅FN₆O 515.29 515.24-14 iPr (S) C₂₉H₃₅FN₆O 503.29 503.2 4-15 —CH₂cPr (S) C₃₀H₃₅FN₆O 515.29515.2 4-16 oxetan-3-yl (S) C₂₉H₃₃FN₆O₂ 517.27 517.2 4-17 phenyl (R)C₃₃H₃₂FN₇O 538.27 538.2 4-18 phenyl (S) C₃₁H₃₂FN₇O 538.27 538.2 4-19 cPr(S) C₂₉H₃₃FN₆O 501.27 501.2 4-20 cPr (R) C₂₉H₃₃FN₆O 501.27 501.2

TABLE 5

Calc Found Ex. No. R⁸ R³, R⁴, R⁵, R⁷ Formula [M + H]⁺ [M + H]⁺ 5-1 —CH₃R³ = —CH₃ C₂₈H₃₅FN₆O 491.29 491.2 R⁴ = —CH₃ R⁷ = —CH₃ 5-2 iPr R³ = —OHC₂₈H₃₃FN₆O₂ 505.27 505 R⁷ = —CH₃ 5-3 —CH₃ R³ = —OH C₂₆H₃₁FN₆O₂ 479.25479 R⁷ = —CH₃ 5-4 —CH₃ R³ = —OH C₂₇H₃₃FN₆O₂ 493.27 493 R⁴ = —CH₃ R⁷ =—CH₃ 5-5 R³ = —OH C₂₅H₂₉FN₆O₂ 465.23 465 R⁴ = —CH₃ 5-6 R⁵ = (S)phenylC₃₀H₃₁FN₆O 511.25 511.1 5-7 R³ = (S)OH C₂₆H₃₁FN₆O₂ 479.25 479 5-8 R³ =(R)OH C₂₆H₃₁FN₆O₂ 479.25 479 5-9 —(CH₂)₂CN R³ = —OH, C₂₈H₃₂FN₇O₂ 518.26518.2 R⁷ = —CH₃ 5-10 —(CH₂)₂OCH₃ R³ = —OH, C₂₈H₃₅FN₆O₃ 523.28 523.2 R⁷ =—CH₃ 5-11 iPr R³ = —OH, C₂₈H₃₅FN₆O₂ 507.28 507.2 R⁷ = —CH₃ 5-12 —C₂H₅ R³= —OH, C₂₇H₃₃FN₆O₂ 493.27 493.2 R⁷ = —CH₃ 5-13 —CH₃ R³ = —CH₃ C₂₇H₃₃FN₆O477.27 477 R⁷ = —CH₃ 5-14 R³ = F C₂₄H₂₆F₂N₆O 453.21 453 5-15 —CH₃ R⁷ =—CH₃ C₂₆H₃₁FN₆O 463.25 463.1 5-16 cPr R⁷ = —CH₃ C₂₈H₃₃FN₆O 489.27 489.25-17 iPr R⁷ = —CH₃ C₂₈H₃₅FN₆O 491.29 491.1 5-18 cBu R⁷ = —CH₃ C₂₉H₃₅FN₆O503.29 503.6 5-19

R⁷ = —CH₃ C₃₀H₃₇FN₆O₂ 533.30 533.6 5-20 tetrahydropyran-4-yl R⁷ = —CH₃C₃₀H₃₇FN₆O₂ 533.30 533.6 5-21 —CH₂CH₂F R⁷ = —CH₃ C₂₇H₃₂F₂N₆O 495.26495.1

TABLE 6

Calc Found Ex. [M + [M + No. R⁸ * Formula H]⁺ H]⁺ 6-1 —CH₃ (S)C₂₇H₃₁FN₆O₂ 491.25 491.1 6-2 —CH₃ (R) C₂₇H₃₁FN₆O₂ 491.25 491.2 6-3—CH₂pheny1 (R) C₃₃H₃₅FN₆O₂ 567.28 567.2 6-4

(R) C₃₀H₃₅FN₆O₃ 547.28 547.2 6-5 —CH₂CH₂OH (R) C₂₈H₃₃FN₆O₃ 521.26 521.3

TABLE 7

Ex. Calc Found No. R^(x1), R^(x2), R^(x3), R^(x4) Formula [M + H]⁺ [M +H]⁺ 7-1 R^(x2) = (R)F C₂₇H₃₀F₂N₆O 493.25 493.2 7-2 R^(x2) = (S)FC₂₇H₃₀F₂N₆O 493.25 493.2 7-3 C₂₇H₃₁FN₆O 475.25 475.2 7-4 R^(x3) andR^(x4) C₂₈H₃₁FN₆O 487.25 487.6 form —CH₂— 7-5 R^(x1) = —CH₂OCH₃C₂₉H₃₅FN₆O₂ 519.28 519.2 7-6 R^(x2) = OH C₂₇H₃₁FN₆O₂ 491.25 491.2 7-7R^(x2) = —C₂H₅ C₂₉H₃₅FN₆O 503.29 503.2 7-8 R^(x2) = phenyl C₃₃H₃₅FN₆O551.29 551.2  7-10 R^(x2) = (S)CH₂OH C₂₈H₃₃FN₆O₂ 505.27 505.2  7-11R^(x2) = —OiPr C₃₀H₃₇FN₆O₂ 533.30 533.2  7-12 R^(x2) = —CH₂OCH₃C₂₉H₃₅FN₆O₂ 519.28 519.2

TABLE 8

Calc Found Ex. No. R⁸ Formula [M + H]⁺ [M + H]⁺ 8-1 C₂₅H₂₇FN₆O 447.22447.2 8-2 —CH₃ C₂₆H₂₉FN₆O 461.24 461.2 8-3 cBu C₂₉H₃₃FN₆O 501.27 501.28-4 —CH₂cPr C₂₉H₃₃FN₆O 501.27 501.2 8-5 iPr C₂₈H₃₃FN₆O 489.27 489.1 8-6tetrahydropyran-4-yl C₃₀H₃₅FN₆O₂ 531.28 531.2 8-7 —CH₂iPr C₂₉H₃₅FN₆O503.29 503.2 8-8 —C₂H₅ C₂₇H₃₁FN₆O 475.25 475.1 8-9 phenyl C₃₀H₃₀FN₇O524.25 524.2  8-10 cPr C₂₈H₃₁FN₆O 487.25 487.2  8-11 oxetan-3-ylC₂₈H₃₁FN₆O₂ 503.25 503.2

TABLE 9

Calc Found Ex. No. R⁸ Formula [M + H]⁺ [M + H]⁺ 9-1 C₂₅H₂₇FN₆O 447.22447.1 9-2 —CH₃ C₂₆H₂₉FN₆O 461.24 461.2 9-3 oxetan-3-yl C₂₈H₃₁FN₆O₂503.25 503.1 9-4 —CH₂iPr C₂₉H₃₅FN₆O 503.29 503.2 9-5

C₂₉H₃₃FN₆O₃S 565.23 565.2 9-6 cBu C₂₉H₃₃FN₆O 501.27 501.3 9-7 —C₂H₅C₂₇H₃₁FN₆O 475.25 475.2 9-8 iPr C₂₈H₃₃FN₆O 489.27 489.2 9-9 —CH₂cPrC₂₉H₃₃FN₆O 501.27 501.2  9-10 tetrahydropyran-4-yl C₃₀H₃₅FN₆O₂ 531.28531.2  9-11 cPr C₂₈H₃₁FN₆O 487.25 487.2  9-12 phenyl C₃₀H₃₀FN₇O 524.25524.2

TABLE 10

Calc Found Ex. No. R⁸ Formula [M + H]⁺ [M + H]⁺ 10-1 iPr C₂₉H₃₅FN₆O503.29 503.2 10-2 —CH₂iPr C₃₀H₃₇FN₆O 517.30 517.2 10-3 —CH₂cPrC₃₀H₃₅FN₆O 515.29 515.2 10-4 —C₂H₅ C₂₈H₃₃FN₆O 489.27 489.2 10-5 cBuC₃₀H₃₅FN₆O 515.29 515.2 10-6 oxetan-3-yl C₂₉H₃₃FN₆O₂ 517.27 517.2 10-7tetrahydropyran-4-yl C₃₁H₃₇FN₆O₂ 545.30 545.2 10-8 —CH₃ C₂₇H₃₁FN₆O475.25 475.2 10-9 cPr C₂₉H₃₃FN₆O 501.27 501.2  10-10 phenyl C₃₁H₃₂FN₇O538.27 538.2

TABLE 11

Calc Found Ex. No. R⁸ Formula [M + H]⁺ [M + H]⁺ 11-1 —CH₃ C₂₈H₃₃FN₆O489.27 489.2 11-2 —C₂H₅ C₂₉H₃₅FN₆O 503.29 503.2 11-3 oxetan-3-ylC₃₀H₃₅FN₆O₂ 531.28 531.2 11-4 —CH₂iPr C₃₁H₃₉FN₆O 531.32 531.2 11-5—CH₂cPr C₃₁H₃₇FN₆O 529.30 529.2 11-6 cBu C₃₁H₃₇FN₆O 529.30 529.2 11-7tetrahydropyran-4-yl C₃₂H₃₉FN₆O₂ 559.31 559.2 11-8 iPr C₃₀H₃₇FN₆O 517.30517.2 11-9 cPr C₃₀H₃₅FN₆O 515.29 515.2  11-10 C₂₇H₃₁FN₆O 475.25 475.2

TABLE 12

Calc Found Ex. No. R^(x1), R^(x2), R^(x3), R^(x4) Formula [M + H]⁺ [M +H]⁺ 12-1 R^(x3) = —CN C₂₇H₂₈FN₇O 486.23 486.1 12-2 R^(x3) = —CN, R^(x4)= —CH₃ C₂₈H₃₀FN₇O 500.25 500.2 12-3 R^(x1) = —CH₃, R^(x2) = —CH₃C₂₈H₃₃FN₆O 489.27 489.2 12-4 R^(x3) = —CH₃ C₂₇H₃₁FN₆O 475.25 475.1 12-5R^(x3) = —CH₃, R^(x4) = —OCH₃ C₂₈H₃₃FN₆O₂ 505.27 505.2 12-6 R^(x3) =—CH₂CH₂OH C₂₈H₃₃FN₆O₂ 505.27 505.2 12-7 R^(x3) = —CH₃, R^(x4) = —CH₃C₂₈H₃₃FN₆O 489.27 489.2 12-8 R^(x3) = —CH₂OC₂H₅ C₂₉H₃₅FN₆O₂ 519.28 519.212-9 R^(x3) = phenyl C₃₂H₃₃FN₆O 537.27 537.2  12-10 R^(x3) = —CH₃,R^(x4) = —OH C₂₇H₃₁FN₆O₂ 491.25 491.2  12-11 C₂₆H₂₉FN₆O 461.24 461.2 12-12 R^(x3) = —CH₂OH C₂₇H₃₁FN₆O₂ 491.25 491.1  12-13 R^(x3) and R^(x4)form C₃₁H₃₇FN₆O 529.30 529.2 —(CH₂)₅—  12-14 R^(x3) and R^(x4) formC₂₈H₃₁FN₆O₂ 503.25 503.2 —CH₂OCH₂—  12-15 R^(x1) = phenyl C₃₂H₃₃FN₆O537.27 536.6

TABLE 13

Calc Found Ex. No. R^(x2), R^(x3), R^(x4) Formula [M + H]⁺ [M + H]⁺ 13-1C₂₈H₃₃FN₆O 489.27 489.2 13-2 R^(x3) = —OH C₂₈H₃₃FN₆O₂ 505.27 505.2 13-3R^(x3) = —OCH₃ C₂₉H₃₅FN₆O₂ 519.28 519.2 13-4 R^(x3) = F C₂₈H₃₂F₂N₆O507.26 507.2 13-5 R^(x3) = —CN C₂₉H₃₂FN₇O 514.27 514.2 13-6 R^(x3) =—CH₂OH C₂₉H₃₅FN₆O₂ 519.28 519.2 13-7 R^(x3) and R^(x4) form C₃₁H₃₇FN₆O529.30 529.3 —(CH₂)₃— 13-8 R^(x2) = —CH₂OH C₂₉H₃₅FN₆O₂ 519.28 519.2 13-9R^(x2) = —OH C₂₈H₃₃FN₆O₂ 505.27 505.2

TABLE 14

Calc Found Ex. No. R⁸ R³ Formula [M + H]⁺ [M + H]⁺ 14-1 —OCH₃C₂₆H₂₉FN₆O₂ 477.23 477 14-2 —CH₃ —OH C₂₆H₂₉FN₆O₂ 477.23 477 14-3 —OHC₂₅H₂₇FN₆O₂ 463.22 463 14-4 —CH₃ —OCH₃ C₂₇H₃₁FN₆O₂ 491.25 490 14-5 —CH₃C₂₆H₂₉FN₆O 461.24 461.1 14-6 —CH₃ F C₂₆H₂₈F₂N₆O 479.23 479 14-7C₂₅H₂₇FN₆O 447.22 447.1 14-8 F C₂₅H₂₆F₂N₆O 465.21 465 14-9 cPrC₂₈H₃₁FN₆O 487.25 487.2  14-10 —(CH₂)₂CN C₂₈H₃₀FN₇O 500.25 500.2  14-11

C₃₀H₃₅FN₆O₂ 531.28 531.3  14-12 cBu C₂₉H₃₃FN₆O 501.27 501.3  14-13—(CH₂)₃OCH₃ C₂₉H₃₅FN₆O₂ 519.28 519.3  14-14 —(CH₂)₂OCH₃ C₂₈H₃₃FN₆O₂505.27 505.2  14-15 oxetan-3-yl C₂₈H₃₁FN₆O₂ 503.25 503.2

TABLE 15

Calc Found Ex. No. R⁸ R³ * Formula [M + H]⁺ [M + H]⁺ 15-1 —CH₃ —OHC₂₇H₃₁FN₆O₂ 491.25 491 15-2 —OH C₂₆H₂₉FN₆O₂ 477.23 477 15-3 (R)C₂₆H₂₉FN₆O 461.24 461.1 15-4 -iPr C₂₉H₃₅FN₆O 503.29 503.6 15-4 cBuC₃₀H₃₅FN₆O 515.29 515.6 15-6

C₃₁H₃₇FN₆O₂ 545.30 545.6 15-7 cPr C₂₉H₃₃FN₆O 501.27 501.2 15-8 —CH₂CH₂FC₂₈H₃₂F₂N₆O 507.26 507.1 15-9 —CH₃ C₂₇H₃₁FN₆O 475.25 475.2  15-10tetrahydropyran-4-yl C₃₁H₃₇FN₆O₂ 545.30 545.2

TABLE 16

Calc Found Ex. No. R⁸ * Formula [M + H]⁺ [M + H]⁺ 16-1 —CH₃ (R)C₂₇H₃₁FN₆O₂ 491.25 491.2 16-2 (R) C₂₆H₂₉FN₆O₂ 477.23 477.2 16-3 (S)C₂₆H₂₉FN₆O₂ 477.23 477.2 16-4 cBu (S) C₃₀H₃₅FN₆O₂ 531.28 531.2 16-5 cPr(S) C₂₉H₃₃FN₆O₂ 517.27 517.3 16-6 iPr (S) C₂₉H₃₅FN₆O₂ 519.28 519.6

TABLE 17

Ex. Calc Found No. R³ R^(x) * Formula [M + H]⁺ [M + H]⁺ 17-1 —OH (S)C₂₇H₃₁FN₆O₂ 491.25 491 17-2 —OH (R) C₂₇H₃₁FN₆O₂ 491.25 491 17-3 —CHF₂C₂₈H₃₁F₃N₆O 525.25 525.2

TABLE 18

Calc Found Ex. No. R⁸ R⁶, R⁷ Formula [M + H]⁺ [M + H]⁺ 18-1 —CH₃ R⁷ =—CH₃ C₂₇H₃₁FN₆O 475.25 475.2 18-2 —CH₃ R⁶ = —CH₃, C₂₈H₃₃FN₆O 489.27 489R⁷ = —CH₃ 18-3 C₂₅H₂₇FN₆O 447.22 447.2

TABLE 19

Calc Found Ex. No. R Formula [M + H]⁺ [M + H]⁺ 19-1

C₂₇H₃₁FN₆O₂ 491.25 491.1 19-2

C₂₉H₃₄F₂N₆O 521.28 521.1

Example 17: Crystalline hydrate of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To a 3 L flask was added NMP (239 mL) and5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol,2 HCl (74.5 g, 165 mmol) with stirring followed by NMP (74 mL). Aceticacid (31.3 mL) was added and the reaction mixture was warmed to 55° C.for 10 min and then cooled to 25° C. 1-methylpiperidin-4-one (61.0 mL,496 mmol) was added in a single portion and the reaction mixture wasstirred at 25° C. for 30 min and cooled to 15° C. Sodiumtriacetoxyborohydride (98 g, 463 mmol) was added and the external jacketwas set to 20° C. after 5 min. After 3 h, ammonium hydroxide (365 mL,5790 mmol) was added dropwise over 45 min maintaining the temperaturebelow 25° C. The reaction mixture was stirred for 1.5 h at 20° C.,forming an off-white slury. Methanol (709 mL) was added and the reactionmixture was stirred slowly overnight at 55° C. Water (1.19 L) was addedover 30 min at 55° C. and the mixture was cooled to 10° C., stirred for2 h, and filtered. The cake was washed with 1:1 MeOH:water (334 mL),dried on the filter for 20 min and at 45° C. under vacuum with nitrogenbleed to provide yellow solids (87 g).

To the solids was added 5% water/acetone (1.5 L) at 55° C. with slowstirring and the reaction mixture was heated at 55° C. for 6 h, cooledto 10° C., filtered, and washed with 5% water/acetone (450 mL). Thesolids were dried overnight at 50° C. under vacuum with nitrogen bleed,equilibrated in air for 20 h, dried in the vacuum oven for 48 h andequilibrated with air to provide the title compound (71.3 g, 91% yield)as a free flowing pale yellow solid. HPLC Method C Retention time 12.29min.

Example 18: Crystalline hydrate of5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To a flask was added5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(45.4 g, 95 mmol) and water (450 mL) and 37% NH₄OH (11.25 mL) and theslurry was stirred for 10 min and filtered. The wet cake was transferredto a 2 L flask and 2.5% water/acetone (900 mL) was added and the slurrywas stirred overnight. Additional water (23 mL) was added and themixture was stirred for 48 h, warmed to 55° C. and stirred at 55° C.overnight. Additional water (69 mL) was added and the slurry was stirredat 25° C. overnight and warmed to 55° C. After 3 h, an additionalportion of water (23 mL) was added, the mixture was cooled to RT,filtered, and washed with 15% water/acetone (250 mL). The solids weredried overnight in a vacuum oven at 50° C. to provide the title compound(32.4 g, 68.3 mmol, 72% yield, 99/2% purity). HPLC Method C Retentiontime 12.27 min. ¹H NMR (400 MHz, Methanol-d4) δ 8.24 (dd, J=8.4, 0.8 Hz,1H), 7.39 (t, J=1.1 Hz, 1H), 7.13 (dd, J=8.4, 1.4 Hz, 1H), 6.93 (d,J=11.7 Hz, 1H), 6.88 (d, J=9.0 Hz, 1H), 3.78 (s, 2H), 3.00 (dd, J=10.0,4.0 Hz, 4H), 2.81 (t, J=5.8 Hz, 2H), 2.68-2.57 (m, 1H), 2.53 (q, J=7.5Hz, 2H), 2.30 (s, 3H), 2.18-2.06 (m, 2H), 1.99 (d, J=11.9 Hz, 2H), 1.75(td, J=12.4, 3.7 Hz, 2H), 1.05 (t, J=7.5 Hz, 3H).

Comparison Example C-1:5-Ethyl-2-fluoro-4-(3-(5-(3-methylcyclobutyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

To a solution of5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolTFA (60 mg, 0.122 mmol) and 3-methylcyclobutane-1-one (51 mg, 0.610mmol) in MeOH (1.221 mL) was added sodium cyanoborohydride (77 mg, 1.221mmol) and the reaction mixture was stirred for 4 h. The reaction mixturewas concentrated by rotary evaporation, dissolved in 2:1 aceticacid:water (1.5 mL) and purified by preparative HPLC to provide the TFAsalt of the title compound (40 mg). (m/z): [M+H]⁺ calcd for C₂₆H₂₈FN₅O446.23 found 446.1.

Using a similar process, substituting the appropriate reagent for3-methylcyclobutane-1-one, the following comparison compounds wereprepared:

Comparison Compounds C-2 to C-4

Ex. Calc Found No. R Formula [M + H]⁺ [M + H]⁺ C-2

C₂₆H₃₀FN₅O 448.24 448.2 C-3

C₂₇H₃₀FN₅O₂ 476.24 476.2 C-4

C₂₈H₃₂FN₅O 474.26 474.2

Examples 19-21: Properties of the Solid Form of the Invention

Samples of the crystalline hydrate5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenolof Example 18 was analyzed by powder X-ray diffraction (PXRD),differential scanning calorimetry (DSC), thermogravimetric analysis(TGA), and dynamic moisture sorption (DMS).

Example 19 Powder X-Ray Diffraction

The powder X-ray diffraction patterns of FIG. 1 was obtained with aBruker D8-Advance X-ray diffractometer using Cu-Kα radiation (λ=1.54051Å) with output voltage of 45 kV and current of 40 mA. The instrument wasoperated in Bragg-Brentano geometry with incident, divergence, andscattering slits set to maximize the intensity at the sample. Formeasurement, a small amount of powder (5-25 mg) was gently pressed ontoa sample holder to form a smooth surface and subjected to X-rayexposure. The samples were scanned in 2θ-2θ mode from 2° to 40° in 2θwith a step size of 0.02° and a scan speed of 0.30° seconds per step.The data acquisition was controlled by Bruker DiffracSuite measurementsoftware and analyzed by Jade software (version 7.5.1). The instrumentwas calibrated with a corundum standard, within ±0.02° two-theta angle.Observed PXRD two-theta peak positions and d-spacings are shown in Table20.

TABLE 20 PXRD Data for the Crystalline Hydrate 2-Theta d(Å) Area A %6.20 14.24 81639 45.70 9.58 9.22 178629 100.00 10.34 8.55 30022 16.8010.65 8.30 12801 7.20 11.54 7.66 27220 15.20 12.77 6.93 27705 15.5013.01 6.80 48785 27.30 13.39 6.61 9261 5.20 16.94 5.23 40031 22.40 17.535.05 83718 46.90 18.67 4.75 9542 5.30 19.28 4.60 152922 85.60 20.02 4.4322391 12.50 20.61 4.31 30308 17.00 21.51 4.13 92875 52.00 22.10 4.0237495 21.00 22.79 3.90 13802 7.70 23.22 3.83 12117 6.80 25.16 3.54 137927.70 28.80 3.10 14487 8.10 29.62 3.01 14810 8.30 30.20 2.96 9709 5.40

Example 20: Thermal Analysis

Differential scanning calorimetry (DSC) was performed using a TAInstruments Model Q-100 module with a Thermal Analyst controller. Datawere collected and analyzed using TA Instruments Thermal Analysissoftware. A sample of each crystalline form was accurately weighed intoa covered aluminum pan. After a 5 minute isothermal equilibration periodat 5° C., the sample was heated using a linear heating ramp of 10°C./min from 0° C. to 250° C. A representative DSC thermogram of the FormI crystalline freebase of the invention is shown in FIG. 2.

Thermogravimetric analysis (TGA) measurements were performed using a TAInstruments Model Q-50 module equipped with high resolution capability.Data were collected using TA Instruments Thermal Analyst controller andanalyzed using TA Instruments Universal Analysis software. A weighedsample was placed onto a platinum pan and scanned with a heating rate of10° C. from ambient temperature to 300° C. The balance and furnacechambers were purged with nitrogen flow during use. A representative TGAtrace of the Form I crystalline freebase of the invention is shown inFIG. 3.

Example 21: Dynamic Moisture Sorption Assessment

Dynamic moisture sorption (DMS) measurement was performed using a VTIatmospheric microbalance, SGA-100 system (VTI Corp., Hialeah, Fla.33016). A weighed sample was used and the humidity was lowest possiblevalue (close to 0% RH) at the start of the analysis. The DMS analysisconsisted of an initial drying step (˜0% RH) for 120 minutes, followedby two cycles of sorption and desorption with a scan rate of 5% RH/stepover the humidity range of 5% RH to 90% RH. The DMS run was performedisothermally at 25° C. A representative DMS trace for the Form Icrystalline freebase of the invention is shown in FIG. 4.

Biological Assays

The compounds of the invention have been characterized in one or more ofthe following biological assays.

Assay 1: Biochemical JAK and Off-target Kinase Assays

A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 andTyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH7.5, 0.01% Brij-35, 10 mM MgCl₂, and 1 mM EGTA). Recombinant GST-taggedJAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained fromLife Technologies.

Serially diluted compounds were pre-incubated with each of the four JAKenzymes and the substrate in white 384-well microplates (Corning) atambient temperature for 1 h. ATP was subsequently added to initiate thekinase reactions in 10 μL total volume, with 1% DMSO. The final enzymeconcentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and0.25 nM respectively; the corresponding Km ATP concentrations used are25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200nM for all four assays. Kinase reactions were allowed to proceed for 1hour at ambient temperature before a 10 μL preparation of EDTA (10 mMfinal concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (LifeTechnologies, 2 nM final concentration) in TR-FRET dilution buffer (LifeTechnologies) was added. The plates were allowed to incubate at ambienttemperature for 1 h before being read on the EnVision reader (PerkinElmer). Emission ratio signals (520 nm/495 nm) were recorded andutilized to calculate the percent inhibition values based on DMSO andbackground controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC₅₀ values were determined from a4-parameter robust fit model with the Prism software (GraphPadSoftware). Results were expressed as pIC₅₀ (negative logarithm of IC50)and subsequently converted to pK_(i) (negative logarithm of dissociationconstant, Ki) using the Cheng-Prusoff equation.

Test compounds having a higher pK_(i) value in each of the four JAKassays show greater inhibition of JAK activity. Compounds of theinvention tested in this assay typically exhibited pK_(i) values betweenabout 9 and about 10.5

A panel of off-target tyrosine kinase assays (Flt3, RET, FGFR2, TrkA,and pDGFRβ) were developed using a similar methodology, with recombinantenzymes obtained from Life Technologies and biotinylated peptidesubstrates synthesized at AnaSpec. All assays were carried out atambient temperature with a final ATP concentration of 100 μM. Detectionreagents, including Eu-anti-phosphotyrosine (pY20) antibody andSureLight APC-SA, were purchased from Perkin Elmer. Emission ratiosignals (665 nm/615 nm) were recorded and utilized for data analysis,and the final results were expressed as pIC₅₀.

Assay 2: Cellular JAKI Potency Assay

The AlphaScreen JAKI cellular potency assay was carried out by measuringinterleukin-13 (IL-13, R&D Systems) induced STAT6 phosphorylation inBEAS-2B human lung epithelial cells (ATCC). The anti-STAT6 antibody(Cell Signaling Technologies) was conjugated to AlphaScreen acceptorbeads (Perkin Elmer), while the anti-pSTAT6 (pTyr641) antibody (CellSignaling Technologies) was biotinylated using EZ-Link Sulfo-NHS-Biotin(Thermo Scientific).

BEAS-2B cells were grown at 37° C. in a 5% CO₂ humidified incubator in50% DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS(Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin (LifeTechnologies), and 2 mM GlutaMAX (Life Technologies). On day 1 of theassay, cells were seeded at a 7,500 cells/well density in whitepoly-D-lysine-coated 384-well plates (Corning) with 25 μL medium, andwere allowed to adhere overnight in the incubator. On day 2 of theassay, the medium was removed and replaced with 12 μL of assay buffer(Hank's Balanced Salt Solution/HBSS, 25 mM HEPES, and 1 mg/ml bovineserum albumin/BSA) containing dose-responses of test compounds.Compounds were serially diluted in DMSO and then diluted another1000-fold in media to bring the final DMSO concentration to 0.1%. Cellswere incubated with test compounds at 37° C. for 1 h, and followed bythe addition of 12 μl of pre-warmed IL-13 (80 ng/ml in assay buffer) forstimulation. After incubating at 37° C. for 30 min, the assay buffer(containing compound and IL-13) was removed, and 10 μl of cell lysisbuffer (25 mM HEPES, 0.1% SDS, 1% NP-40, 5 mM MgCl2, 1.3 mM EDTA, 1 mMEGTA, and supplement with Complete Ultra mini protease inhibitors andPhosSTOP from Roche Diagnostics). The plates were shaken at ambienttemperature for 30 min before the addition of detection reagents. Amixture of biotin-anti-pSTAT6 and anti-STAT6 conjugated acceptor beadswas added first and incubated at ambient temperature for 2 h, followedby the addition of streptavidin conjugated donor beads (Perkin Elmer).After a minimum of 2 h incubation, the assay plates were read on theEnVision plate reader. AlphaScreen luminescence signals were recordedand utilized to calculate the percent inhibition values based on DMSOand background controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC₅₀ values were determined from a4-parameter robust fit model with the Prism software. Results wereexpressed as the negative logarithm of the IC₅₀ value, pIC₅₀.

Test compounds having a higher pIC₅₀ value in this assay show greaterinhibition of IL-13 induced STAT6 phosphorylation. Compounds of theinvention tested in this assay typically exhibited pIC₅₀ values betweenabout 7.5 and about 8.5.

Assay 3: Cytotoxicity Assay

A CellTiter-Glo luminescent cell viability/cytotoxicity assay wascarried out in BEAS-2B human lung epithelial cells (ATCC) under thenormal growth condition.

Cells were grown at 37° C. in a 5% CO₂ humidified incubator in 50%DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS(Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin (LifeTechnologies), and 2 mM GlutaMAX (Life Technologies). On day 1 of theassay, cells were seeded at a 500 cells/well density in white 384-welltissue culture plates (Corning) with 25 μL medium, and were allowed toadhere overnight in the incubator. On day 2 of the assay, 5 μL of mediumcontaining dose-responses of test compounds was added, and incubated at37° C. for 48 h. 30 μl of CellTiter-Glo detection solution (Promega) wassubsequently added, mixed on an orbital shaker for 5 min, and incubatedfor additional 10 min before being read on the EnVision reader.Luminescence signals were recorded and percent DMSO control values werecalculated.

For dose-response analysis, percent DMSO control data were plotted vs.compound concentrations to derive dose-response curves by lineconnecting each data point. The concentration at which each curvecrosses the 15% inhibition threshold is defined as CC₁₅. Results wereexpressed as the negative logarithm of the CC₁₅ value, pCC₁₅.

It is expected that test compounds exhibiting a lower pCC₁₅ value inthis assay have less likelihood to cause cytotoxicity. Compounds of theinvention tested in this assay typically exhibited pCC₁₅ values betweenless than 5 and about 6.6.

In Vitro Assay Results

All of the compounds of Examples 1 to 16 and Tables 1 to 19 were testedin one or more of the assays described above. In the following tables,for the JAK1, JAK 2, JAK3, and TYK2 enzyme assays, A represents a pK_(i)value ≥10 (K_(i)≤0.1 nM), B represents a pK_(i) value between 9 and 10(K_(i) between 1 nM and 0.1 nM), C represents a pK_(i) value between 9and 9.5 (K_(i) between 1 nM and 0.32 nM), and D represents a pK_(i)value between 8.5 and 9 (K_(i) between 32 nM and 1 nM). For the BEAS-2Bcell potency assay, A represents a pIC₅₀ value ≥8 (IC₅₀≤10 nM) and Brepresents a pIC₅₀ value between 7.4 and 8 (IC₅₀ between 40 nM and 10nM). The following compounds were tested in all of the in vitro assaysdescribed above.

Example JAK 1 JAK 2 JAK 3 Tyk 2 BEAS-2B Number (pK_(i)) (pK_(i))(pK_(i)) (pKi) pIC₅₀ 1 A A A B A 2 A A A B B 3 A A B B A 4 A A B B A 5 AA B C A 6 A A B B A 7 A A B C A 8 A A B B A 9 A A B C A 10 A A B B A 11B A B C A 12 A A B C A 13 A A B C A 14 A A B B A 15 A A B C A 16 A A A BA 1-1 A A B C B 1-3 A A A C B 1-6 A A A C A 1-8 A A A C A 2-3 A A B B B 3-30 A A B B A 4-1 B B C D B 4-2 A A A C B 7-3 A A A B A 9-1 A A B C B9-2 A A A C A 14-7  A A A C B

It was observed that JAK 1 enzyme potency was predictive of cellularpotency in the BEAS-2B3 assay. Therefore all of the remaining compoundswere tested in the JAK1 enzyme assay and the cellular assay andexhibited a pKi enzyme value between 9 and 10.5 and a BEAS-2B3 pIC₅₀value between 7.4 and 8.5 with the exception of compounds 3-32, 4-8.4-16. and 8-11, which exhibited JAK inhibition at a pK_(i) enzyme valuebetween 8.5 and 9 and cellular potency between 6 and 7.4.

Assay 4: Pharmacokinetics in Plasma and Lung in Mouse

Plasma and lung levels of test compounds and ratios thereof weredetermined in the following manner. BALB/c mice from Charles RiverLaboratories were used in the assay. Test compounds were individuallyformulated in 20% propylene glycol in pH 4 citrate buffer at aconcentration of 0.2 mg/mL and 50 uL of the dosing solution wasintroduced into the trachea of a mouse by oral aspiration. At varioustime points (typically 0.167, 2, 6, 24 hr) post dosing, blood sampleswere removed via cardiac puncture and intact lungs were excised from themice. Blood samples were centrifuged (Eppendorf centrifuge, 5804R) for 4minutes at approximately 12,000 rpm at 4° C. to collect plasma. Lungswere padded dry, weighed, and homogenized at a dilution of 1:3 insterile water. Plasma and lung levels of test compound were determinedby LC-MS analysis against analytical standards constructed into astandard curve in the test matrix. A lung to plasma ratio was determinedas the ratio of the lung AUC in μg hr/g to the plasma AUC in μg hr/mL,where AUC is conventionally defined as the area under the curve of testcompound concentration vs. time. Compounds of the invention exhibitedexposure in lung from one to two orders of magnitude greater thanexposure in plasma in mouse. All of the compounds profiled in this assayexhibited a half-life between about 5 and about 12 hours.

Assay 5: Murine (Mouse) Model of IL-13 Induced pSTAT6 Induction in LungTissue

Il-13 is an important cytokine underlying the pathophysiology of asthma(Kudlacz et al. Eur. J Pharmacol, 2008, 582, 154-161). IL-13 binds tocell surface receptors activating members of the Janus family of kinases(JAK) which then phosphorylate STAT6 and subsequently activates furthertranscription pathways. In the described model, a dose of IL-13 wasdelivered locally into the lungs of mice to induce the phosphorylationof STAT6 (pSTAT6) which is then measured as the endpoint.

Adult balb/c mice from Harlan were used in the assay. On the day ofstudy, animals were lightly anesthetized with isoflurane andadministered either vehicle or test compound (0.5 mg/mL, 50 μL totalvolume over several breaths) via oral aspiration. Animals were placed inlateral recumbency post dose and monitored for full recovery fromanesthesia before being returned to their home cage. Four hours later,animals were once again briefly anesthetized and challenged with eithervehicle or IL-13 (0.03 μg total dose delivered, 50 μL total volume) viaoral aspiration before being monitored for recovery from anesthesia andreturned to their home cage. One hour after vehicle or IL-13administration, lungs were collected for both pSTAT6 detection using ananti-pSTAT6 ELISA (rabbit mAb capture/coating antibody; mouse mAbdetection/report antibody: anti-pSTAT6-pY641; secondary antibody:anti-mouse IgG-HRP) and analyzed for total drug concentration asdescribed above in Assay 4.

Selected compounds of the invention were tested in the assay. Activityin the model is evidenced by a decrease in the level of pSTAT6 presentin the lungs of treated animals at 5 hours compared to the vehicletreated, IL-13 challenged control animals. The difference between thecontrol animals which were vehicle-treated, IL-13 challenged and and thecontrol animals which were vehicle-treated, vehicle challenged dictatedthe 0% and 100% inhibitory effect, respectively, in any givenexperiment. Exemplary compounds of the invention were tested in theassay, and exhibited inhibition of STAT6 phosphorylation at 4 hoursafter IL-13 challenge as documented below. The compounds 1-15 and 3-1were noted as exceptions under the conditions of the assay.

Confirming the relevance of the JAK-STAT pathway in airway inflammation,compounds which have demonstrated in vivo target engagement in theIL13-induced pSTAT6 mouse model are subsequently tested and proven to beefficacious in a mouse model of allergen-induced eosinophilicinflammation.

In Vivo Assay Results

Selected compounds of the invention were characterized in both thepharmacokinetic assay (Assay 4) and pharmacodynamic assay (Assay 5). Agood correlation was observed between test compound concentration inlung determined in the pharmacokinetic assay and in the pharmacodynamicassay at a similar time points post dosing. Observation of significantcompound concentration in the mouse lung in the pharmacodynamic assayconfirmed that the observed inhibition of IL-13 induced pSTAT6 inductionwas a result of the activity of the test compound.

In the following table, for the ratio of lung exposure to plasmaexposure (Assay 4), A denotes a ratio >100, B denotes a ratio between 50and 100, and C denotes a ratio between 10 and 50. For the percentinhibition of IL-13 induced pSTAT6 induction (Assay 5), Arepresents >65% inhibition, B represents between 50% and 65% inhibitionand C represents between 33% and 50% inhibition.

Lung to Plasma pSTAT6 Example ratio inhibition Number Assay 4 Assay 5 1B A 2 C C 3 C A 4 B A 5 C A 6 B B 7 B A 8 A B 9 B B 10 B C 11 A C 12 C C13 A C 14 A A 15 A B 1-7  B C 1-11 C C 1-18 B C 3-33 A C 14-7   A B

Assay 6: Murine Model of Alternaria alternata-Induced EosinophilicInflammation of the Lung

Airway eosinophilia is a hallmark of human asthma. Alternaria alternatais a fungal aeroallergen that can exacerbate asthma in humans andinduces eosinophilic inflammation in the lungs of mice (Havaux et al.Clin Exp Immunol. 2005, 139(2):179-88). In mice, it has beendemonstrated that alternaria indirectly activates tissue resident type 2innate lymphoid cells in the lung, which respond to (e.g. IL-2 and IL-7)and release JAK-dependent cytokines (e.g. IL-5 and IL-13) and coordinateeosinophilic inflammation (Bartemes et al. J Immunol. 2012,188(3):1503-13).

Seven- to nine-week old male C57 mice from Taconic were used in thestudy. On the day of study, animals were lightly anesthetized withisoflurane and administered either vehicle or test compound (0.1-1.0mg/mL, 50 μL total volume over several breaths) via oropharyngealaspiration. Animals were placed in lateral recumbency post dose andmonitored for full recovery from anesthesia before being returned totheir home cage. One hour later, animals were once again brieflyanesthetized and challenged with either vehicle or alternaria extract(200 ug total extract delivered, 50 μL total volume) via oropharyngealaspiration before being monitored for recovery from anesthesia andreturned to their home cage. Forty-eight hours after alternariaadministration, bronchoalveolar lavage fluid (BALF) was collected andeosinophils were counted in the BALF using the Advia 120 HematologySystem (Siemens).

Selected compounds of the invention were tested in this alternariaassay. Activity in the model is evidenced by a decrease in the level ofeosinophils present in the BALF of treated animals at forty-eight hourscompared to the vehicle treated, alternaria challenged control animals.Data are expressed as percent inhibition of the vehicle treated,alternaria challenged BALF eosinophils response. To calculate percentinhibition, the number of BALF eosinophils for each condition isconverted to percent of the average vehicle treated, alternariachallenged BALF eosinophils and subtracted from one-hundred percent.Exemplary compounds of the invention were tested in the assay andexhibited inhibition of BALF eosinophil counts at forty-eight hoursafter alternaria challenge as documented below.

In Vivo Assay Results

All of the compounds tested demonstrated a range of inhibition (60%-98%)of alternaria-induced BALF eosinophils. The following table reflects themaximum statistically significant percent inhibition of the vehicletreated, alternaria challenged level of eosinophil induction.

Per cent Inhibition of Alternaria-induced BALF Example NumberEosinophils  1 98  2 93  3 61  6 88  7 96  8 60 11 71 13 82

Characterization of Comparison Compounds

The correspondence between the comparison compounds and a compound ofthe invention is illustrated below.

Compound of the Invention Comparison Compound Example No. R Example No.R  1

C-1

 7

C-2

13

C-3

 6

C-4

The comparison compounds were characterized in the JAK1 enzyme assay,the BEAS-2B3 cellular assay and the pSTAT6 inhibition pharmacodynamicassay. Comparison compounds C-1, C-2, C-3, and C-4 were 2-fold, 3-fold,6-fold, and 2.5-fold less potent, respectively, than the correspondingcompound of the invention in the enzyme assay and 6-fold, 6-fold,3-fold, and 6-fold less potent, respectively, than the correspondingcompound of the invention in the BEAS-2B cellular assay. The comparisoncompounds did not exhibit pSTAT6 inhibition in the pharmacodynamicassay. Also, in that assay, the compounds did not show significant lungconcentration. The lung concentration observed for comparison compoundsC-1, C-2, C-3, and C-4 was smaller than that observed for thecorresponding compound of the invention by a factor of 36, 52, 13, and23, respectively.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

1.-31. (canceled)
 32. A pharmaceutical composition comprising a compoundof formula (I):

wherein: X is a group of formula (II):

n is 0 or 1; R¹ is hydrogen or C₁₋₃alkyl; R² is hydrogen or C₁₋₃alkyl;R³ is hydrogen or C₁₋₃alkyl; or R² and R³ taken together formC₂₋₄alkylene; or, when n is 1, R³ is selected from hydrogen, —OH,—OC₁₋₃alkyl, halo, —C(O)OC₁₋₃alkyl, and C₁₋₃alkyl, wherein C₁₋₃alkyl isoptionally substituted with —OH; R⁴ is hydrogen or C₁₋₃alkyl; R⁵ isselected from hydrogen, C₁₋₃alkyl, —C(O)OC₁₋₃alkyl, and phenyl; or whenn is 1, R² and R⁵ taken together form C₁₋₃alkylene; R⁶ is hydrogen orC₁₋₃alkyl; R⁷ is hydrogen or C₁₋₃alkyl, or when n is 0, R² and R⁷ takentogether form C₁₋₃alkylene, or R⁴ and R⁷ taken together formC₂₋₄alkylene or C₁alkylene-O—C₂alkylene; or when n is 1, R² and R⁷ takentogether form C₂alkylene, optionally substituted with C₁₋₃alkyl orR^(x), or R⁴ and R⁷ taken together form C₁₋₃alkylene or —O—C₂alkylene;R⁸ is selected from (a) hydrogen, (b) methyl, optionally substitutedwith —CN, phenyl or C₃₋₆cycloalkyl; (c) C₂₋₆alkyl, wherein C₂₋₆alkyl isoptionally substituted with one or two substituents selected from —OH,—OC₁₋₃alkyl, —CN, —SC₁₋₃alkyl, phenyl, C₃₋₆cycloalkyl, halo, andoptionally, in addition with two substituents on a single carbon atomtaken together to form C₂₋₃alkylene; (d) C₃₋₆cycloalkyl, whereinC₃₋₆cycloalkyl is optionally substituted with —OH, —CN, —OC₁₋₃alkyl, orC₁₋₃alkyl, wherein C₁₋₃alkyl is optionally substituted with —OC₁₋₃alkylor with one or two halo, (e) oxetanyl, (f) tetrahydropyranyl, (g)tetrahydrothiophenyl 1,1-dioxide, and (h) phenyl, or R⁷ and R⁸ takentogether form C₃₋₅alkylene or C₂alkylene-O—C₂alkylene; whereinC₃₋₅alkylene is optionally substituted with one or two R^(x); R^(x) isselected from —OH, —CN, —OC₁₋₃alkyl, halo, phenyl, and C₁₋₃alkyl whichis optionally substituted with —OC₁₋₃alkyl or —OH, or two substituentsR^(x) taken together form C₁₋₅alkylene or —CH₂OCH₂—, or when n is 1 andR² and R⁷ taken together form C₂alkylene, R⁴ and a substituent R^(x) onC₂alkylene taken together form C₂alkylene; provided that twosubstituents R^(x) on the same carbon atom are not both fluoro, andprovided that when R^(x) is attached to a carbon atom adjacent to anitrogen atom, R^(x) is not —OH, —OC₁₋₃alkyl, or halo; or apharmaceutically-acceptable salt thereof, and apharmaceutically-acceptable carrier; wherein the pharmaceuticalcomposition is a nebulizer composition.
 33. The nebulizer composition ofclaim 32, wherein the composition comprises about 0.05 μg/mL to about 20mg/mL of the compound of formula (I).
 34. The nebulizer composition ofclaim 32, wherein the composition provides about 0.1 mg to about 4 mg ofthe compound of formula (I) per dose.
 35. The nebulizer composition ofclaim 32, wherein the composition comprises 3% by weight of glycerol.36. The nebulizer composition of claim 32, wherein the composition has apH between about 3.0 to about 8.0.
 37. The nebulizer composition ofclaim 32, wherein the composition has a pH between about 3.5 to about5.5.
 38. The nebulizer composition of claim 32, wherein the compositionis administered with a nebulizer inhaler.
 39. The nebulizer compositionof claim 32, wherein the compound of formula (I) is a compound of theformula:

or a pharmaceutically acceptable salt thereof.
 40. The nebulizercomposition of claim 32, wherein the compound of formula (I) is acompound of the formula:

or a pharmaceutically acceptable salt thereof.
 41. A method of treatinga respiratory disease modulated by Janus kinases in a subject havingsaid disease, the method comprising administering to the subject apharmaceutical composition of claim
 32. 42. The method of claim 41,wherein composition of claim 32 comprises the compound:

or a pharmaceutically acceptable salt thereof.
 43. The method of claim41, wherein composition of claim 32 comprises the compound:

or a pharmaceutically acceptable salt thereof.
 44. The method of claim41, wherein the respiratory disease is asthma, chronic obstructivepulmonary disease, cystic fibrosis, pneumonitis, idiopathic pulmonaryfibrosis, acute lung injury, acute respiratory distress syndrome,bronchitis, emphysema or bronchiolitis obliterans.
 45. The method ofclaim 41, wherein the respiratory disease is chronic obstructivepulmonary disease.
 46. The method of claim 41, wherein the respiratorydisease is asthma.
 47. The method of claim 41, wherein the respiratorydisease is bronchiolitis obliterans.